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Title:
ETHODS OF DETECTING ALDEHYDES IN SAMPLES
Document Type and Number:
WIPO Patent Application WO/2022/122846
Kind Code:
A1
Abstract:
The present disclosure provides methods for derivatizing and detecting aldehydes and ketones formed as degradation products of fatty acids and fatty acid esters. The methods form stable derivatives of the aldehydes and ketones that allow for subsequent separation and detection.

Inventors:
SCHROETER NINA ARIANE (DE)
KOULOV ATANAS (CH)
MAHLER HANNS-CHRISTIAN (DE)
JAHN MICHAEL (DE)
Application Number:
PCT/EP2021/084838
Publication Date:
June 16, 2022
Filing Date:
December 08, 2021
Export Citation:
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Assignee:
LONZA AG (CH)
International Classes:
G01N31/22; C07C29/16
Foreign References:
US20060105464A12006-05-18
Other References:
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Attorney, Agent or Firm:
GREINER, Elisabeth (DE)
Download PDF:
Claims:
Claims

1. A method of derivatizing a C1-C20 aldehyde in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises a derivatizing agent and an acid, and ii. wherein the mixture provides a derivatization reaction between the C1-C20 aldehyde and the derivatizing agent to form a derivatized aldehyde; and b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction.

2. A method of detecting a C1-C20 aldehyde in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises a derivatizing agent and an acid; and ii. wherein the mixture provides a derivatization reaction between the C1-C20 aldehyde and the derivatizing agent to form a derivatized aldehyde; b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction, and c) detecting the derivatized aldehyde.

A method of detecting a C1-C20 aldehyde in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises an organic solvent, a derivatizing agent and an acid; ii. wherein the sample comprises an aqueous solvent; and iii. wherein the mixture provides a derivatization reaction between the C1-C20 aldehyde and the derivatizing agent to form a derivatized aldehyde; b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction, and wherein the quenched mixture forms an organic phase and an aqueous phase; c) isolating the organic phase of the quenched mixture containing the derivatized aldehyde; and d) detecting the derivatized aldehyde in the organic phase.

4. The method of any preceding claim, wherein the C1-C20 aldehyde is an unsaturated, a mono-unsaturated or a poly-unsaturated aldehyde.

5. The method of any preceding claim, wherein the C1-C20 aldehyde is optionally substituted with a hydroxyl, methyl, ethyl, halogen, carbonyl, benzyl or combinations thereof or with one or more hydroxyl substituents.

6. The method of any preceding claim, wherein the C1-C20 aldehyde is a C6-C16 aldehyde, a C8-C10 aldehyde or a nonenal, optionally wherein the nonenal is a 2-nonenal, a 4-nonenal or a 6- nonenal.

The method of any of claims 1-4, wherein the C1-C20 aldehyde is 4-hydroxynonenal, trans-2-nonenal, 2-undecenal, 2-decenal, crotonaldehyde, benzaldehyde, 4-hydroxy-2-hexenal, 2- 4-nonadienal or 2-4-decadienal, 4-oxononenaL, formaldehyde, acetaldehyde, butanal, pentanal, hexanal, heptanal, octanal, 2-octenal, nonanal, 2-nonenal, decanal, undecanal, dodecanal, tridecanal, acrolein, or benzaldehyde.

8. The method of any preceding claim, wherein the derivatizing agent is a substituted hydrazine or Purpald (4-Amino-3-hydarzino-5-mercapto-l,2,4-triazol, ammonia and acetylacetone or dimedone, or an O-Alkylated hydroxylamine selected from O- methylhydroxylamine, O-benzylhydroxylamine, O-(p-nitrobenzyl)hydroxylamine or O- pentafluorobenzylhydroxylamine (PFBOA).

9. The method of claim 8, wherein the substituted hydrazine is

(i) an aryl hydrazine;

(ii) a phenyl hydrazine, optionally wherein the phenyl hydrazine is substituted with one or more of nitro, hydroxyl, alkyl, halogen or combinations thereof or wherein the phenyl hydrazine is a dihydrophenylhydrazine; or (iii) 2,4-dinitrophenylhydrazine, 2,5-dinitrophenylhydrazine, 2,6-dinitrophenylhydrazine, 3,4- dinitrophenylhydrazine, or 3,5-dinitrophenylhydrazine, pentafluorophenylhydrazine, 2- chlorophenylhydrazine, 2,4-dichlorophenylhydrazine, (cyclohexanedione) N-methyl- benzothiazolon-(2)-hydrazone (MBTH), 4-nitrophenylhydrazine, 1 -methyl- 1 -(2,4- dinitrophenyl)-hydrazine (MDNPH) l-Dimethylaminonaphthalene-5-sulfonylhydrazide (Dansyl hydrazine DNSH), Hydrazine reagent based on the benzooxadiazole backbone, 2-diphenylacetyl- 1,3 -indandione- 1 -hydrazone (DAIH) or 2,4,6-trichlorophenylhydrazine (TCPH).

10. The method of any preceding claim, wherein the derivatization solution comprises about 0.005M to about IM, about 0.02 M to about 0.1M, or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 M derivatizing agent.

11. The method of any preceding claim, wherein the mixture comprises about 0.0005M to about IM, about 0.01 M to about 0.1M, or about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 M derivatizing agent.

12. The method of any preceding claim, wherein the acid is a weak acid, optionally wherein the weak acid is phosphoric acid, formic acid, acetic acid, benzoic acid, oxalic acid, hydrofluoric acid, nitrous acid or sulfiirous acid.

13. The method of any of claims 1, 2 and 4-12, wherein the derivatization solution further comprises an organic solvent, optionally wherein the organic solvent is acetonitrile, acetone, 1,4- dioxane, tetrahydrofuran 1 -propanol, 2-propanol, methanol, ethanol, 1 -butanol, formamide, N- methylformamide, propylenecarbonate, N,N-dimethyl formamide or N,N-dimethyl sulfoxide.

14. The method of any of claims 3-13, wherein a ratio of organic solvent to acid in the derivatization solution is from about 1:5 to about 5:1 (v/v), optionally about 3:1 (v/v).

15. The method of any preceding claim, wherein the sample is an aqueous sample, optionally comprising a protein, optionally a therapeutic protein, or wherein the sample is a lab culture, food preparation, cosmetic, water sample or pharmaceutical preparation. 16. The method of any preceding claim, wherein the sany le further comprises

(i) a fatty acid.

(ii) an unsaturated fatty acid; or

(iii) an emulsifier, optionally wherein the emulsifier is a polysorbate, optionally wherein the polysorbate is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80.

17. The method of any preceding claim, wherein the C1-C20 aldehyde in the sample is leached from a packaging, a manufacturing apparatus, or an administration device, optionally

(i) wherein the packaging comprises an ampule, vial, bottle, container, screw cap, carton, stopper, or tubing;

(ii) wherein the packaging comprises a polymer, resin, metal, or glass;

(iii) wherein the container comprises polyethylene, polyvinyl chloride, or a plastic additive, optionally wherein the plastic additive in the container is epoxidized soybean oil;

(iv) wherein the administration device is a syringe, a dropper, a spoon, or a dosing cup; or

(v) wherein the sample is obtained by storing an aqueous solution or organic solvent in the packaging, the manufacturing apparatus, or the administration device for an extended period of time, optionally wherein the extended period of time is one day to 1 year.

18. The method of any preceding claim, wherein the ratio of derivatization solution to sany le is from about 1:10 to about 10:1 (v/v), optionally about 1:1 (v/v).

19. The method of any preceding claim, wherein the pH of the mixture after the derivatization reaction is less than about 2.0, or less than about 1.0.

20. The method of any preceding claim, wherein, upon dissociation, the base comprises an anion selected from fluoride, sulfate, hydrogen phosphate, acetate, chloride, nitrate, bromide, chlorate, iodide, perchlorate or thiocyanate and/or

74 a cation selected from ammonium, potassium, sodium, lithium, magnesium calcium or guanidium.

21. The method of any preceding claim, wherein the base is a strong base, optionally sodium hydroxide, potassium hydroxide, barium hydroxide, cesium hydroxide, strontium hydroxide, calcium hydroxide, lithium hydroxide or rubidium hydroxide and/or wherein the base has a concentration of about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0 or 15.0 M prior to addition to the mixture.

22. The method of any preceding claim, wherein the ratio of mixture to base in the quenched mixture is about 20:1 to about 1:5 (v/v).

23. The method of any preceding claim, wherein the pH of the quenched mixture is greater than about 1.0, greater than about 2.0; greater than about 5.0; or about 5.5.

24. The method of any of claims 2-23, wherein the detecting comprises liquid chromatography, mass spectrometry, or combinations thereof, optionally wherein the detecting further comprises measuring the UV absorbance of the derivatized aldehyde.

Description:
METHODS OF DETECTING ALDEHYDES IN SAMPLES

FIELD OF THE INVENTION

[0001] The present disclosure provides for methods of derivatizing and detecting a C 1 -C 20 aldehyde in a sample, such as a sample containing degradation products of fatty acids or fatty acid esters.

BACKGROUND

[0002] Surfactants such as polysorbates are frequently used in foods, cosmetics and pharmaceuticals. For example, polysorbates are frequently used in preparations of therapeutic proteins to stabilize the protein against interfacial stress and surface absorption. These surfactants can also undergo oxidative degradation and form potentially toxic aldehydes.

[0003] Fatty acids, such as unsaturated fatty acids and similar compounds, and fatty acid esters can undergo oxidative degradation leading to the formation of aldehydes. In some embodiments, these aldehydes can be potentially toxic. Often aldehydes formed from unsaturated fatty acids or esters thereof have been found to be difficult to detect in samples containing other degradation products. Current methods for detecting degradation products of fatty acids or esters thereof often use conditions under which certain degradation products are not stable long enough to be detected. For example, degradation products from these methods may form adducts that cannot be properly resolved using chromatography and/or mass spectrometry methods. Additionally, it may be necessary to identify aldehydes which may leach out from a packaging, a container or other parts of the composition.

SUMMARY OF THE INVENTION

[0004] The present disclosure is directed to a method of derivatizing a C 1 -C 20 aldehyde in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises a derivatizing agent and an acid, and ii. wherein the mixture provides a derivatization reaction between the C 1 -C 20 aldehyde and the derivatizing agent to form a derivatized aldehyde; and b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction. [0005] The present disclosure is also directed to a method of detecting a C 1 -C 20 aldehyde in a sample, the method coirprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises a derivatizing agent and an acid; and ii) wherein the mixture provides a derivatization reaction between the C 1 -C 20 aldehyde and the derivatizing agent to form a derivatized aldehyde; b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction, and c) detecting the derivatized aldehyde.

[0006] The present disclosure is further directed to a method of detecting a C 1 -C 20 aldehyde in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises an organic solvent, a derivatizing agent and an acid; ii. wherein the sample comprises an aqueous solvent; and iii wherein the mixture provides a derivatization reaction between the C 1 -C 20 aldehyde and the derivatizing agent to form a derivatized aldehyde; b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction, and wherein the quenched mixture forms an organic phase and an aqueous phase; c) isolating the organic phase of the quenched mixture containing the derivatized aldehyde; and d) detecting the derivatized aldehyde in the organic phase.

[0007] In embodiments of the methods herein, the C 1 -C 20 aldehyde is an unsaturated aldehyde. In embodiments, the C 1 -C 20 aldehyde is a mono-unsaturated aldehyde. In embodiments, the C 1 -C 20 aldehyde is a poly-unsaturated aldehyde. In embodiments, the C 1 -C 20 aldehyde is optionally substituted with a hydroxyl, methyl, ethyl, halogen, carbonyl, benzyl or combinations thereof In embodiments, the C 1 -C 20 aldehyde is optionally substituted with one or more hydroxyl substituents.

[0008] In embodiments of the methods herein, the C 1 -C 20 aldehyde is a C 6 -C 16 aldehyde. In embodiments, the C 1 -C 20 aldehyde is a C 8 -C 10 aldehyde. In embodiments, the C 1 -C 20 aldehyde is a nonenal. In embodiments, the nonenal is a 2-nonenal, a 4-nonenal or a 6-nonenaL

[0009] In embodiments of the methods herein, the C 1 -C 20 aldehyde is 4-hydroxynonenal, trans- 2-nonenal, 2-undecenal, 2-decenal, crotonaldehyde, benzaldehyde, 4-hydroxy-2-hexenal, 2-4- nonadienal or 2-4-decadienal, 4-oxononenal, formaldehyde, acetaldehyde, butanal, pentanal, hexanal, heptanal, octanal, 2-octenal, nonanal, 2-nonenal, decanal, undecanal, dodecanal, tridecanal, acrolein or benzaldehyde. In embodiments, the C 1 -C 20 aldehyde is 4-hydroxynonenal.

[0010] In embodiments of the methods herein, the derivatizing agent is a substituted hydrazine. In embodiments, the substituted hydrazine is an aryl hydrazine. In embodiments, the substituted hydrazine is a phenyl hydrazine. In embodiments, the phenyl hydrazine is substituted with one or more of nitro, hydroxyl, alkyl, halogen or combinations thereof. In embodiments, the phenyl hydrazine is substituted with one or more nitro. In embodiments, the phenyl hydrazine is a dihydrophenylhydrazine. In embodiments, the substituted hydrazine is 2.4- dinitrophenylhydrazine, 2,5 -dinitrophenylhydrazine, 2,6-dinitrophenylhydrazine, 3.4- dinitrophenylhydrazine, or 3,5-dinitrophenylhydrazine, pentafluorophenylhydrazine, 2- chlorophenylhydrazine, 2,4-dichlorophenylhydrazine, (cyclohexanedione) N-methyl- benzothiazolon-(2)-hydrazone (MBTH), 4-nitrophenylhydrazine, 1 -methyl- 1 -(2, 4-dinitrophenyl)- hydrazine (MDNPH) l-Dimethylaminonaphthalene-5-sulfonylhydrazide (Dansyl hydrazine DNSH), Hydrazine reagent based on the benzooxadiazole backbone, 2-diphenylacetyl-l,3- indandione-1 -hydrazone (DAIH) or 2,4,6-trichlorophenylhydrazine (TCPH).

[0011] In embodiments of the methods herein, the derivatizing agent is Purpald (4-Amino-3- hydarzino-5-mercapto-l,2,4-triazol, ammonia and acetylacetone or dimedone, or an O- Alkylated hydroxylamine selected from O-methylhydroxylamine, O-benzylhydroxylamine, O-(p- nitrobenzyl)hydroxylamine or O- pentafluorobenzylhydroxylamine (PFBOA).

[0012] In embodiments of the methods herein, the derivatization solution comprises about 0.005M to about IM derivatizing agent. In embodiments, the derivatization solution comprises about 0.02 M to about 0.1M derivatizing agent. In embodiments, the derivatization solution comprises about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 M derivatizing agent.

[0013] In embodiments of the methods herein, the mixture comprises about 0.0005M to about IM derivatizing agent. In embodiments, the mixture conyrises about 0.01 M to about 0.1M derivatizing agent. In embodiments, the mixture conyrises about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 M derivatizing agent. [0014] In embodiments of the methods herein, the acid is a weak acid. In embodiments, the weak acid is phosphoric acid, formic acid, acetic acid, benzoic acid, oxalic acid, hydrofluoric acid, nitrous acid or sulfurous acid. In embodiments, the weak acid is phosphoric acid.

[0015] In embodiments of the methods herein, the derivatization solution further comprises an organic solvent. In embodiments, a ratio of organic solvent to acid in the derivatization solution is from about 1:5 to about 5:1 (v/v). In embodiments, the ratio of organic solvent to acid in the derivatization solution is about 3:1 (v/v). In embodiments, the organic solvent is acetonitrile, acetone or 1,4-dioxane, tetrahydrofuran 1 -propanol, 2 -propanol, methanol, ethanol, 1 -butanol, formamide, N-methylformamide, propylenecarbonate, N,N-dimethyl formamide or N,N-dimethyl sulfoxide.

[0016] In embodiments of the methods herein, the sample is an aqueous sample. In embodiments, the sample is a lab culture, food preparation, cosmetic, water sample or pharmaceutical preparation. In embodiments, the sample is an aqueous sample comprising a protein. In embodiments, the protein is a therapeutic protein.

[0017] In embodiments of the methods herein, the sample further comprises a fatty acid. In embodiments, the sample further comprises an unsaturated fatty acid. In embodiments, the sample further comprises an emulsifier. In embodiments, the emulsifier is a polysorbate. In embodiments, the polysorbate is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80. In embodiments, the sample further comprises a solubilizer, e.g., a solubilizing agent, e.g., an organic solvent.

[0018] In embodiments of the methods herein, the ratio of derivatization solution to sample is from about 1 : 10 to about 10: 1 (v/v). In embodiments, the ratio of derivatization solution to sample is about 1:1 (v/v).

[0019] In embodiments of the methods herein, the pH of the mixture after the derivatization reaction is less than about 2.0. In embodiments, the pH of the mixture after the derivatization reaction is less than about 1.0.

[0020] In embodiments of the methods herein, upon dissociation, the base comprises an anion selected from fluoride, sulfate, hydrogen phosphate, acetate, chloride, nitrate, bromide, chlorate, iodide, perchlorate or thiocyanate. In embodiments, upon dissociation, the base comprises a cation selected from ammonium, potassium, sodium, lithium, magnesium calcium or guanidium.

[0021] In embodiments of the methods herein, the base is a strong base. In embodiments, the strong base is sodium hydroxide, potassium hydroxide, barium hydroxide, cesium hydroxide, strontium hydroxide, calcium hydroxide, lithium hydroxide or rubidium hydroxide. In embodiments, the strong base is sodium hydroxide. In embodiments, the base has a concentration of about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0 or 15.0 M prior to addition to the mixture.

[0022] In embodiments of the methods herein, the ratio of mixture to base in the quenched mixture is about 20:1 to about 1:5 (v/v). In embodiments, the pH of the quenched mixture is greater than about 1.0. In embodiments, the pH of the quenched mixture is greater than about 2.0. In embodiments, the pH of the quenched mixture is greater than about 5.0. In embodiments, the pH of the quenched mixture is about 5.5.

[0023] In embodiments of the methods herein having a detecting step, the detecting comprises liquid chromatography, mass spectrometry, or combinations thereof. In embodiments of these methods, the detecting further comprises measuring the UV absorbance of the derivatized aldehyde.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following drawings form part of the present specification and are included to further demonstrate exemplary embodiments of certain aspects of the present invention.

[0025] FIGS. 1A-1D show the chemical structures as described. FIG. 1A) shows polysorbate (PS; simplified, with m = 5 for PS20, and m =8 with one unsaturated unit for PS80). The sum of the ethylene oxide moieties (w, x, y, and z) is in total about 20). FIG. 1B) shows linoleic acid. FIG. 1C) shows 4-Hydroxynonenal (HNE). FIG. 1D) shows butylated hydroxyl toluene (BHT).

[0026] FIG. 2 is a schematic of potential mechanisms for lipid peroxidation of n-6 polyunsaturated fatty acids (PUFAs), such as linoleic acid, resulting in HNE formation. [0027] FIG. 3 is a schematic of the derivatization reaction of carbonyls with 2,4-dinitrophenyl hydrazine (DNPH) forming hydrazones.

[0028] FIG. 4 is a plot of Monitored UV absorbance signals at 364 nm of repeated injections of hexanal-DNPH (circle), HNE-DNPH (cross) and trans-2-nonenal-DNPH (diamond) in aqueous PS80 solution. The ordinate shows intensity of the UV signal in arbitrary units (AU) and the abscissa the number of the repeated injection.

[0029] FIG. 5 shows an overlay of UV chromatograms obtained at an absorbance wavelength of 364 nm of the HNE-DNPH reaction mixture after an incubation time of 20 min (black trace), 12 h (light grey trace), and 48 h (dark grey trace). The ordinate shows intensity of the UV signal in AU and the abscissa the retention time in min. Compound 1 represents HNE-DNPH hydrazone including the peak shoulder, which likely corresponds to the second, less favored stereoisomer, compound 2 the double DNPH-HNE adduct detected after 12 h, and compound 3 follow-up products detected after 48 h.

[0030] FIGS. 6A and 6B show plots of monitored UV signal at an absorbance wavelength of 364 nm of A) the HNE-DNPH hydrazone peak, and B) the double adduct. Samples were re- injected every two hours. The signals obtained in the sample containing 0 M NaOH are displayed by black filled circles, 1 M NaOH by white filled diamonds, and 10 M NaOH by stars.

[0031] FIGS. 7 A and 7B are graphs showing MRM signal intensity upon varying the collision energy (CE) (FIG. 7 A) and declustering potential DP (FIG. 1B) monitoring the transitions m/z 335 > 167 (black columns) and 335 > 163 (grey columns). Average values of duplicate injections with standard deviations are shown.

[0032] FIG. 8 is a plot showing overlay of LC-MRM (transition 335 > 167) chromatograms of worked-up PS80 control sample not containing spiked HNE (black trace, 0 ppb HNE) and the standard sample containing HNE at a concentration of 0.5 ppb (grey trace, 0.5 ppb HNE).

[0033] FIG. 9 is a plot of the average areas of triplicate injections with standard deviations of HNE-DNPH standards ranging from 2 to 75 ppb. In addition, standards containing 5 ppb, 20 ppb and 50 ppb HNE were worked-up in triplicate to determine the work-up precision and are displayed separately by three independent average areas. The accuracy of all standards investigated was ± 20% of the target spiking level.

[0034] FIG. 10 is a plot of the determination of BHT concentration in low peroxide PS80 by standard addition using LC-UV analysis at an absorbance wavelength at 278 nm. Solving the linear equation resulted in a concentration of ~600 ppb, and taking into account the dilution factor before analysis (10 3 ) yielded a concentration of 0.06% (w/w) in the bulk PS.

[0035] FIGS. 11A-C are plots of the kinetics of HNE formation from 10% (w/v) aqueous PS80 solutions under various oxidative stress conditions. Stress panel: FIG. 11 A) Fenton’s reagent at RT, FIG. 11B) 1.5 mM AAPH at 40°C/75% RH, FIG. 11C) air exposure at 40°C/75% RH. Black columns represent not stabilized PS80 (J.T. Baker grade), light grey columns represent Lonza’s PS80 (0.02% (w/w) BHT), blacked framed white columns represent commercially available low peroxide PS80 (0.006% (w/w) BHT). Control represents samples frozen at -20°C for the time course of the kinetic study. Average values of triplicate injections with standard deviations are shown.

[0036] FIG. 12 is a schematic illustration of the apparatuses used in the simulated administration leachable study.

[0037] FIG: 13. LC-UV -MS results of the leachable detected in the extracts of the administration sets.

[0038] FIG. 14: Overlay of the obtained LC-MRM signals of the HNE-DNPH hydrazone in the administration set extracts and controls is shown.

[0039] FIG. 15: Primary HNE-re leasing material screening study. UV signals obtained in the MeOH extracts of the individual administration materials at an absorbance wavelength at 220 nm corresponding to reference standards are shown.

[0040] FIG. 16: Confirmation of suspected HNE -releasing materials. Overlay of obtained HNE- DNPH hydrazone MRM signals using the transition 335 > 167 in the derivatized extracts of the three suspected materials. DETAILED DESCRIPTION

[0041] The present disclosure provides a method of derivatizing a C 1 -C 20 aldehyde in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises a derivatizing agent and an acid, and ii. wherein the mixture provides a derivatization reaction between the C 1 -C 20 aldehyde and the derivatizing agent to form a derivatized aldehyde; and b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction.

[0042] As used herein, “a” or “an” may mean one or more. As used herein, when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein, “another” or “a further” may mean at least a second or more.

[0043] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the method/device being employed to determine the value, or the variation that exists among the study subjects. Typically, the term “about” is meant to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% or higher variability, depending on the situation. In some embodiments, one of skill in the art will understand the level of variability indicated by the term “about,” due to the context in which it is used herein. It should also be understood that use of the term “about” also includes the specifically recited value.

[0044] The use of the term “or” in the claims is used to mean “and/or,” unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”

[0045] As used herein, the terms “comprising” (and any variant or form of comprising, such as “comprise” and “comprises”), “having” (and any variant or form of having, such as “have” and “has”), “including” (and any variant or form of including, such as “includes” and “include”) or “containing” (and any variant or form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0046] The use of the term “for example” and its corresponding abbreviation “e.g.” (whether italicized or not) means that the specific terms recited are representative examples and embodiments of the disclosure that are not intended to be limited to the specific examples referenced or cited unless explicitly stated otherwise.

[0047] As used herein, “between” is a range inclusive of the ends of the range. For example, a number between x and y explicitly includes the numbers x and y, and any numbers that frill within x and y.

[0048] In embodiments, the present disclosure provides methods of derivatizing and detecting aldehydes or ketones in samples.

[0049] In embodiments, the present disclosure provides a method of derivatizing an aldehyde or ketone in a sample. In embodiments, the disclosure provides a method of derivatizing an aldehyde or ketone in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises a derivatizing agent and an acid, and ii. wherein the mixture provides a derivatization reaction between the aldehyde or ketone and the derivatizing agent to form a derivatized aldehyde or ketone; and b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction.

[0050] In embodiments, the present disclosure provides a method of derivatizing an aldehyde in a sample. In embodiments, the disclosure provides a method of derivatizing a C 1 -C 20 aldehyde in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises a derivatizing agent and an acid, and ii. wherein the mixture provides a derivatization reaction between the C 1 -C 20 aldehyde and the derivatizing agent to form a derivatized aldehyde; and b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction.

[0051] In embodiments, the derivatization of the aldehyde or ketone in the samples makes it easier to detect the aldehyde or ketone. In embodiments, the derivatization of the aldehyde or ketone stabilizes the aldehyde or ketone in the sample to prevent it from degrading, forming adducts or otherwise undergoing a chemical change. In embodiments, the aldehyde or ketone is stabilized for a period of at least about 1, 2, 5, 10, 12, 15, 20, 24, 36, 48, 60, 72, 84, 96 or more hours. In embodiments, the aldehyde or ketone is stabilized for a period of at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days. In embodiments, the aldehyde or ketone is stabilized for a period of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks. In embodiments, the aldehyde or ketone is stabilized for a period of 1, 2, 3, 4, 5, 6 or more months.

[0052] Without wishing to be bound by theory, in specific embodiments, the methods herein improve the stability and detectability of aldehydes and ketones in treated samples in part due to a salting-out effect on the sample. In another non-limiting theory, in some embodiments, the methods described herein provide a stabilizing effect of the neutralization reaction on the hydrazones. In specific embodiments, this salting-out effect is the result of adjustments in the pH and/or salt concentration of the mixture. In embodiments, the methods cause the salting-out of the organic solvent used during derivatization, leading to improved stability of the aldehydes and ketones. This improved stability may allow for improved detectability of the aldehydes and ketones as it prevents the formation of adducts and other derivation products that are difficult to resolve and detect using methods such as liquid chromatography and mass spectrometry. In some embodiments, because the sample matrix is cleaner, the MRM signal intensity increases, resulting in less ion-suppression and a stronger signal.

[0053] In embodiments, the present disclosure provides a method of detecting an aldehyde or ketone in a sample. In embodiments, the disclosure provides a method of detecting an aldehyde or ketone in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises a derivatizing agent and an acid; and ii. wherein the mixture provides a derivatization reaction between the aldehyde or ketone and the derivatizing agent to form a derivatized aldehyde or ketone; b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction, and c) detecting the derivatized aldehyde or ketone.

[0054] In embodiments, the present disclosure provides a method of detecting an aldehyde in a sample. In embodiments, the disclosure provides a method of detecting a C 1 -C 20 aldehyde in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises a derivatizing agent and an acid; and ii. wherein the mixture provides a derivatization reaction between the C 1 -C 20 aldehyde and the derivatizing agent to form a derivatized aldehyde; b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction, and c) detecting the derivatized aldehyde.

[0055] In embodiments, the present disclosure also provides a method of detecting an aldehyde or ketone in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises an organic solvent, a derivatizing agent and an acid; ii. wherein the sample comprises an aqueous solvent; and iii. wherein the mixture provides a derivatization reaction between the aldehyde or ketone and the derivatizing agent to form a derivatized aldehyde or ketone; b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction, and wherein the quenched mixture forms an organic phase and an aqueous phase; c) isolating the organic phase of the quenched mixture containing the derivatized aldehyde or ketone; and d) detecting the derivatized aldehyde or ketone in the organic phase.

[0056] In embodiments, the present disclosure also provides a method of detecting a C 1 -C 20 aldehyde in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises an organic solvent, a derivatizing agent and an acid; ii. wherein the sample comprises an aqueous solvent; and iii. wherein the mixture provides a derivatization reaction between the C 1 -C 20 aldehyde and the derivatizing agent to form a derivatized aldehyde; b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction, and wherein the quenched mixture forms an organic phase and an aqueous phase; c) isolating the organic phase of the quenched mixture containing the derivatized aldehyde; and d) detecting the derivatized aldehyde in the organic phase.

[0057] In embodiments of the methods herein, the aldehyde is a C 1 -C 20 aldehyde. In embodiments of the methods herein, the ketone is a C 1 -C 20 ketone. In embodiments, the aldehyde or ketone can be unsaturated, partially saturated or fully saturated. In embodiments, the C 1 -C 20 aldehyde is an unsaturated aldehyde. In embodiments, the C 1 -C 20 aldehyde is a mono-unsaturated aldehyde, e.g., an aldehyde having a single carbon-carbon double bond. In embodiments, the C 1 -C 20 aldehyde is a poly-unsaturated aldehyde, e.g., an aldehyde having two or more carbon-carbon double bonds. In embodiments, the C 1 -C 20 ketone is an unsaturated aldehyde. In embodiments, the C 1 -C 20 ketone is a mono-unsaturated ketone, e.g., a ketone having a single carbon-carbon double bond. In embodiments, the C 1 -C 20 ketone is a poly-unsaturated ketone, e.g., a ketone having two or more carbon-carbon double bonds.

[0058] In embodiments of the methods herein, the C 1 -C 20 aldehyde is substituted with one or more substituent groups. In embodiments, the C 1 -C 20 aldehyde is optionally substituted with a hydroxyl, methyl, ethyl, halogen, carbonyl, benzyl or combinations of these substituents. In embodiments, the C 1 -C 20 aldehyde is optionally substituted with one or more hydroxyl substituents. In embodiments of the methods herein, the C 1 -C 20 ketone is substituted with one or more substituent groups. In embodiments, the C 1 -C 20 ketone is optionally substituted with a hydroxyl, methyl, ethyl, halogen, carbonyl, benzyl or combinations of these substituents. In embodiments, the C 1 -C 20 ketone is optionally substituted with one or more hydroxyl substituents.

[0059] The carbon chain of the aldehyde or ketone can have varying lengths. In embodiments, the C 1 -C 20 aldehyde is a C 6 -C 16 aldehyde. In embodiments, the C 1 -C 20 aldehyde is a C 8 -C 10 aldehyde. In embodiments, the C 1 -C 20 aldehyde is a C 6 -C 16 aldehyde. In embodiments, the C 1 -C 20 aldehyde is a C 2 -C 19 , a C 3 -C 18 , a C 4 -C 17 , a C 5 -C 16 , a C 6 -C 15 , a C 7 -C 14 , a C 8 -C 13 , or a C 9 -C 12 aldehyde. In embodiments, the aldehyde is a C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16, C 17 , C 18 , C 19 or C 20 aldehyde. In embodiments, the aldehyde is a straight chain aldehyde. In embodiments, the aldehyde is a branched chain aldehyde.

[0060] In embodiments, the C 1 -C 20 ketone is a C 6 -C 16 ketone. In embodiments, the C 1 -C 20 ketone is a C 8 -C 10 ketone. In embodiments, the C 1 -C 20 ketone is a C 6 -C 16 ketone. In embodiments, the C 1 - C 20 ketone is a C 2 -C 19 , a C 3 -C 18 , a C 4 -C 17 , a C 5 -C 16 , a C 6 -C 15 , a C 7 -C 14 , a C 8 - C 13 or a C 9 -C 12 ketone. In embodiments, the ketone is a C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , C 15 , C 16, C 17 , C 18 , C 19 or C ketone. In embodiments, the ketone is a straight chain ketone. In embodiments, the ketone is a branched chain ketone.

[0061] In embodiments of the methods herein, the C 1 -C 20 aldehyde is a nonenal In embodiments, the nonenal is a 2-nonenal, a 4-nonenal or a 6-nonenal. In embodiments, the C 1 -C 20 aldehyde is 4-hydroxynonenal, trans-2-nonenal, 2-undecenal, 2-decenal, crotonaldehyde, benzaldehyde, 4-hydroxy-2-hexenal 2-4-nonadienal or 2-4-decadienal, 4-oxononenal, formaldehyde, acetaldehyde, butanal, pentanal, hexanal, heptanal, octanal, 2-octenal, nonanal, 2- nonenal, decanal, undecanal, dodecanal, tridecanal, acrolein or benzaldehyde. In embodiments, the C 1 -C 20 aldehyde is 4-hydroxynonenal (HNE).

[0062] In embodiments of the methods herein, the C 1 -C 20 ketone is acetone, 2-pentanone, 2- hexanone, 2-heptanone, 2-octanone, 2-octen-4-one, 2-nonanone, 2-decanone, 2-undecanone or 2- dodecanone.

[0063] In embodiments of the methods herein, a derivatizing agent (also known as a derivatizing reagent) is used to derivatize the aldehyde or ketone. In embodiments, the derivatizing agent is a substituted hydrazine. In embodiments, the substituted hydrazine is an aryl hydrazine. In embodiments, the substituted hydrazine is a phenyl hydrazine. In embodiments, the phenyl hydrazine is substituted with one or more of nitro, hydroxyl, alkyl, halogen or combinations thereof. In embodiments, the phenyl hydrazine is substituted with one or more nitro. In embodiments, the phenyl hydrazine is a dihydrophenylhydrazine.

[0064] In embodiments of the methods herein where a substituted hydrazine is used as a derivatization agent, the substituted hydrazine is 2,4-dinitrophenylhydrazine, 2,5- dinitrophenylhydrazine, 2,6-dinitrophenylhydrazine, 3 ,4-dinitrophenylhydrazine, or 3,5- dinitrophenylhydrazine, pentafluorophenylhydrazine, 2-chlorophenylhydrazine, 2,4- dichlorophenylhydrazine, (cyclohexanedione) N-methyl-benzothiazolon-(2)-hydrazone (MBTH), 4-nitrophenylhydrazine, 1 -methyl- 1 -(2, 4-dinitrophenyl)-hydrazine (MDNPH) 1-

Dimethylaminonaphthalene-5-sulfonylhydrazide (Dansyl hydrazine DNSH), Hydrazine reagent based on the benzooxadiazole backbone, 2-diphenylacetyl-l,3-indandione-l -hydrazone (DAIH) or 2,4,6-trichlorophenylhydrazine (TCPH).

[0065] In other embodiments of the methods herein, the derivatizing agent is Purpald (4-Amino- 3-hydarzino-5-mercapto-l,2,4-triazol, ammonia and acetylacetone or dimedone, or O-Alkylated hydroxylamines such as O-methylhydroxylamine, O-benzylhydroxylamine, O-(p- nitrobenzyl)hydroxylamine or O- pentafluorobenzylhydroxylamine (PFBOA).

[0066] In embodiments of the methods herein, the derivatization solution comprises about 0.005M to about IM derivatizing agent. In embodiments, the derivatization solution comprises about 0.02 M to about 0.1M derivatizing agent. In embodiments, the derivatization solution comprises about 0.01 M to about 0.75M derivatizing agent. In embodiments, the derivatization solution comprises about 0.01 M to about 0.5M derivatizing agent. In embodiments, the derivatization solution comprises about 0.025 M to about 0.25M derivatizing agent. In embodiments, the derivatization solution comprises about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 M derivatizing agent. In embodiments, the derivatization solution comprises about 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1.0 M derivatizing agent.

[0067] In embodiments of the methods herein where the derivatization agent is a substituted hydrazine, the derivatization solution comprises about 0.005M to about IM substituted hydrazine. In embodiments, the derivatization solution comprises about 0.02 M to about 0.1M substituted hydrazine. In embodiments, the derivatization solution comprises about 0.01 M to about 0.75M substituted hydrazine. In embodiments, the derivatization solution comprises about 0.01 M to about 0.5M substituted hydrazine. In embodiments, the derivatization solution comprises about 0.025 M to about 0.25M substituted hydrazine. In embodiments, the derivatization solution comprises about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 M substituted hydrazine. In embodiments, the derivatization solution comprises about 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1.0 M substituted hydrazine.

[0068] In embodiments of the methods herein where the derivatization agent is dihydrophenylhydrazine, the derivatization solution comprises about 0.005M to about IM dihydrophenylhydrazine. In embodiments, the derivatization solution comprises about 0.02 M to about 0. IM dihydrophenylhydrazine. In embodiments, the derivatization solution comprises about 0.01 M to about 0.75M dihydrophenylhydrazine. In embodiments, the derivatization solution comprises about 0.01 M to about 0.5M dihydrophenylhydrazine. In embodiments, the derivatization solution comprises about 0.025 M to about 0.25M dihydrophenylhydrazine. In embodiments, the derivatization solution comprises about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 M dihydrophenylhydrazine. In embodiments, the derivatization solution comprises about 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1.0 M dihydrophenylhydrazine. [0069] In embodiments of the methods herein where the derivatization agent is 2,4- dihydrophenylhydrazine, the derivatization solution comprises about 0.005M to about IM 2,4- dihydrophenylhydrazine. In embodiments, the derivatization solution comprises about 0.02 M to about 0.1M 2,4-dihydrophenylhydrazine. In embodiments, the derivatization solution comprises about 0.01 M to about 0.75M 2,4-dihydrophenylhydrazine. In embodiments, the derivatization solution comprises about 0.01 M to about 0.5M 2,4-dihydrophenylhydrazine. In embodiments, the derivatization solution comprises about 0.025 M to about 0.25M 2,4-dihydrophenylhydrazine. In embodiments, the derivatization solution comprises about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 M 2,4-dihydrophenylhydrazine. In embodiments, the derivatization solution comprises about 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1.0 M 2,4-dihydrophenylhydrazine.

[0070] The amount of derivatization agent added can be adjusted in order to obtain a specific concentration of derivatization agent in the mixture with the sample. In embodiments, the mixture comprises about 0.0005M to about IM derivatizing agent. In embodiments, the mixture coirprises about 0.01 M to about 0.1M derivatizing agent, hi embodiments, the mixture comprises about 0.001 M to about 0.75M derivatizing agent. In embodiments, the mixture comprises about 0.005 M to about 0.5M derivatizing agent. In embodiments, the mixture comprises about 0.005 M to about 0.25M derivatizing agent. In embodiments, the mixture comprises about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 M derivatizing agent. In embodiments, the mixture comprises about 0.001, 0.002, 0.0025, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009 or 0.01 M derivatizing agent.

[0071] In embodiments where the derivatization agent is a substituted hydrazine, the mixture comprises about 0.0005M to about IM substituted hydrazine. In embodiments, the mixture comprises about 0.01 M to about 0.1M substituted hydrazine. In embodiments, the mixture comprises about 0.001 M to about 0.75M substituted hydrazine. In embodiments, the mixture comprises about 0.005 M to about 0.5M substituted hydrazine. In embodiments, the mixture comprises about 0.005 M to about 0.25M substituted hydrazine. In embodiments, the mixture comprises about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 M substituted hydrazine. In embodiments, the mixture comprises about 0.001, 0.002, 0.0025, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009 or 0.01 M substituted hydrazine. [0072] In embodiments of the methods herein, the derivatization solution comprises an acid. In embodiments, the acid is a weak acid. In embodiments, the weak acid is phosphoric acid, formic acid, acetic acid, benzoic acid, oxalic acid, hydrofluoric acid, nitrous acid or sulfurous acid. In embodiments, the weak acid is phosphoric acid.

[0073] In embodiments, the pH of the derivatization solution is from about 1 to about 6.5. In embodiments, the pH of the derivatization solution is from about 2 to about 6. In embodiments, the pH of the derivatization solution is from about 3 to about 5. In embodiments, the pH of the derivatization solution is from about 1 to 6, 1 to 5.5, 1 to 5.0, 1 to 4.5, 1 to 4.0, 1 to 3.5, or 1 to 3.0. In embodiments, the pH of the derivatization solution is from about 4 to about 6. In embodiments, the pH of the derivatization solution is from about 2 to about 5. In embodiments, the pH of the derivatization solution is from about 1 to about 3. In embodiments, the pH of the derivatization solution is about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5 about 6.0 or about 6.5.

[0074] In embodiments of the methods herein, the derivatization solution comprises an organic solvent. In embodiments, the ratio of organic solvent to acid in the derivatization solution is from about 1:5 to about 5:1 (v/v). In embodiments, the ratio of organic solvent to acid in the derivatization solution is from about 1 : 10 to about 10: 1 (v/v). In embodiments, the ratio of organic solvent to acid in the derivatization solution is from about 1:7 to about 7:1 (v/v). In embodiments, the ratio of organic solvent to acid in the derivatization solution is from about 1:3 to about 3:1 (v/v). In embodiments, the ratio of organic solvent to acid in the derivatization solution is from about 1:2 to about 2:1 (v/v). In embodiments, the ratio of organic solvent to acid in the derivatization solution is about 3:1 (v/v). In embodiments, the ratio of organic solvent to acid in the derivatization solution is about 2:1 (v/v). In embodiments, the ratio of organic solvent to acid in the derivatization solution is about 1:1 (v/v).

[0075] Where the derivatization solution comprises an organic solvent, in embodiments, the organic solvent can be acetonitrile, acetone, 1,4-dioxane, tetrahydro furan 1 -propanol, 2-propanol, methanol, ethanol, 1 -butanol, formamide, N-methylformamide, propylenecarbonate, N,N- dimethyl formamide or N,N-dimethyl sulfoxide. In embodiments, the organic solvent is acetonitrile. [0076] In embodiments, the methods herein are used to analyze a sample suspected of containing fatty acid degradation products. The samples can be obtained during processing of pharmaceutical products (including therapeutic proteins, nucleic acids and other biomolecules), cosmetic products, food products and drink products.

[0077] In embodiments, the samples can include stored pharmaceutical products, e.g., pharmaceutical products stored at least 1 day, 1 week, 1 month, 3 months, 6 months, 1 year or longer after manufacturing. In embodiments, the methods herein can be used to determine whether the aldehyde or ketone level is the pharmaceutical product is suitable to still be used, e.g., the aldehyde or ketone level does not exceed a predetermined level. In certain embodiments, the predetermined level is established by the manufacturers of the pharmaceutical product and/or governmental agency such as the United States Food and Drug Agency, the European Medicines Agency or Japanese Pharmaceuticals and Medical Devices Agency, or other acceptable scientific standard, such as the United States Pharmacopeia (USP), the European Pharmacopeia (EP) of the Japanese Pharmacopeia (JP).

[0078] The samples can also be obtained from medical laboratories, waste treatment plants, ground water sources. The samples can also be obtained from animal sources, such as mammalian sources, including humans. Samples obtained from animal sources include fluid and tissue samples. In embodiments, the sample is an aqueous sample. In embodiments, the sample is a lab culture, food preparation, cosmetic, water sample or pharmaceutical preparation. In some embodiments, the sample is from material testing, e.g., the sample is taken to see if an aldehyde has leached from a container, e.g., a storage container, or a part of a container, e.g., a top or a stopper, etc.

[0079] In embodiments, the sample is an aqueous sample conyrising a protein. In embodiments, the protein is a therapeutic protein. In embodiments, the sample is obtained during manufacturing of the therapeutic protein or the sample is obtained from the final formulation of the therapeutic protein. In these embodiments, the sample may thus contain excipients commonly used in formulations of therapeutic proteins, including buffers, emulsifiers, stabilizers, pH adjusting agents, salts, lubricants, anti-aggregation agents and solubilizers. In embodiments, the sample further comprises a solubilizer, e.g., a solubilizing agent, e.g., an organic solvent. In some embodiments, the solubilizer may cause or accelerate the rate at which an aldehyde, e.g., HNE, is leached from a container or packaging.

[0080] In embodiments of the methods herein, the sample comprises a fatty acid. In embodiments, the sample comprises an unsaturated fritty acid. In embodiments, the sample comprises a polyunsaturated fritty acid. In embodiments, the sample comprises a monounsaturated fatty acid. In embodiments, the sample comprises a saturated fritty acid.

[0081] In some embodiments, the C 1 -C 20 aldehyde in the sample is leached from a packaging, a manufacturing apparatus, or an administration device. Thus, the methods as described herein can be used to determine the existence and the concentration of an aldehyde which has leached from a packaging, a manufacturing apparatus, or an administration device. In some embodiments, the packaging, a manufacturing apparatus, or an administration device is used in the manufacturing, storage or administration of a food, cosmetic, or pharmaceutical product, e.g., a therapeutic protein. In some embodiments, the packaging comprises an ampule, vial, bottle, container, screw cap, carton, stopper or tubing. In some embodiments, the packaging comprises a polymer, resin, metal, or glass. In some embodiments, the container comprises polyethylene, polyvinyl chloride, or a plastic additive. In some embodiments, the plastic additive in the container is epoxidized soybean oil. In some embodiments, the administration device is a syringe, a dropper, a spoon, or a dosing cup. The term packaging can refer to individual components or the sum of packaging components that together contain, protect, and/or enclose the sample. This includes primary packaging components and secondary packaging components. Packaging can include any single part of a packaging system, e.g., containers (e.g., ampules, vials, bottles), container liners (e.g., tube liners), closures (e.g., screw caps, stoppers), closure liners, stopper overseals, container inner seals, administration ports (e.g., on large-volume parenterals (LVPs)), overwraps, administration accessories, and container labels. In some embodiments, the package is or may be in direct contact with the sample. The manufacturing apparatus includes any apparatus which is in contact with the sample during the manufacturing process, including, growth containers, storage containers, lines, ports, etc.

[0082] In some embodiments, the rate of leaching of the aldehyde, e.g., HNE, from the packaging, manufacturing apparatus, or administration device can be dependent on time, i.e., how long the sample is in contact with the packaging, manufacturing apparatus, or administration device. In some embodiments, the rate of leaching can be dependent on the content of the sample, e.g., the solvent or carrier, the temperature, or the age of the packaging, manufacturing apparatus, or administration device. In some embodiments, the rate of leaching can be dependent on humidity, radiation levels (e.g., used in sterilization or from sunlight), or other environmental factors. In some embodiments, the rate of leaching can be dependent on a combination or two or more factors. Due to the potential for an aldehyde to leach from a packaging, manufacturing apparatus, or administration device and contaminate any sample placed in the packaging, manufacturing apparatus, or administration device, in some embodiments, the disclosure provides a method to determine the acceptability of a packaging, manufacturing apparatus, or administration device. For example, in some embodiments, the disclosure provides a method to determine the presence or concentration of an aldehyde which has leached from a packaging, manufacturing apparatus, or administration device, the method comprising (i) placing a sample in a packaging, manufacturing apparatus, or administration device for an extended period of time, (ii) combining the sample with a derivatization solution to form a mixture, wherein the derivatization solution comprises a derivatizing agent and an acid, and, wherein the mixture provides a derivatization reaction between the C 1 -C 20 aldehyde and the derivatizing agent to form a derivatized aldehyde, (iii) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction, and (iv) measuring for the presence and/or concentration of the aldehyde.

[0083] In some embodiments, the disclosure provides a method to determine the suitability of a a packaging, manufacturing apparatus, or administration device to store a sample, the method comprising (i) storing a packaging, manufacturing apparatus, or administration device for an extended period of time after manufacturing, (ii) placing a solvent in the packaging, manufacturing apparatus, or administration device to solubilize any aldehyde which has leached from the packaging, manufacturing apparatus, or administration device, (iii) combining the solvent with a derivatization solution to form a mixture, wherein the derivatization solution comprises a derivatizing agent and an acid, and, wherein the mixture provides a derivatization reaction between the aldehyde and the derivatizing agent to form a derivatized aldehyde, (iv) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction, and (v) measuring for the presence and/or concentration of the aldehyde, thereby determining whether packaging, manufacturing apparatus, or administration device is suitable for storing the sample. [0084] In some embodiments, the sample is obtained by storing an aqueous solution or organic solvent in the packaging, the manufacturing apparatus, or the administration device for an extended period of time. In some embodiments, the extended period of time is one day to 1 year, 1 week to 6 months, or 1 month to 6 months. The skilled artisan can adjust conditions, e.g., temperature, pressure, humidity, etc., to accelerate the leaching of the aldehyde from the container before measurement. In some embodiments, an aqueous solution, e.g., water, a buffer system, a protein product, etc, may be stored in the packaging, the manufacturing apparatus, or the administration device for an extended period of time, and the method of the present invention can be used to determine the existence and/or the amount of aldehyde which leaches from the packaging. In some embodiments, different solvents can used, e.g., an oil or an organic solvent, may be stored in the packaging, the manufacturing apparatus, or the administration device for an extended period of time, and the method of the present invention can be used to determine the existence and/or the amount of aldehyde which leaches from the packaging in the solvent.

[0085] In embodiments of the methods herein, the sample comprises an emulsifier. In embodiments, the emulsifier is a polysorbate. In embodiments, the polysorbate is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80.

[0086] In embodiments of the methods herein, the amount of derivatization solution and amount of sample used to make the mixture can be adjusted. In embodiments, the ratio of derivatization solution to sample is from about 1:10 to about 10:1 (v/v). In embodiments, the ratio of derivatization solution to sample is from about 1 :7 to about 7:1 (v/v). In embodiments, the ratio of derivatization solution to sample is from about 1 :5 to about 5 : 1 (v/v). In embodiments, the ratio of derivatization solution to sample is from about 1 :3 to about 3 : 1 (v/v). In embodiments, the ratio of derivatization solution to sample is from about 1 :2 to about 2: 1 (v/v). In embodiments, the ratio of derivatization solution to sample is about 1:1 (v/v).

[0087] In embodiments of the methods herein, the acid in the derivatization solution that is added lowers the pH of the mixture. In embodiments, the pH of the mixture after the derivatization reaction is less than about 5.5. In embodiments, the pH of the mixture after the derivatization reaction is less than about 5.0. In embodiments, the pH of the mixture after the derivatization reaction is less than about 4.0. In embodiments, the pH of the mixture after the derivatization reaction is less than about 3.0. In embodiments, the pH of the mixture after the derivatization reaction is less than about 2.0. In embodiments, the pH of the mixture after the derivatization reaction is less than about 1.0.

[0088] As used herein, a "base" is substance that reacts with acids and is capable of increasing the pH of a solution to which it is added. When dissolved in an aqueous solution, a base dissociates into ions, i.e., cations and anions.

[0089] In embodiments of the methods herein, a base is added to quench the derivatization reaction. As used herein, the term "quench" refers to the deactivation of the derivatization reaction by increasing the pH and/or increasing the concentration of ions in the mixture.

[0090] In embodiments, the base comprises molecular entities that dissociate when added to solution to form ions. In embodiments, upon dissociation, the base forms at least one ion chosen from the lyotropic series (also known as the Hofrneister series). The lyotropic series of ions comprises both anions and cations that are able to cause a salting-out of compounds in solution. In embodiments, upon dissociation, the base comprises an anion selected from fluoride, sulfate, hydrogen phosphate, acetate, chloride, nitrate, bromide, chlorate, iodide, perchlorate or thiocyanate. In embodiments, upon dissociation, the base comprises a cation selected from ammonium, potassium, sodium, lithium, magnesium, barium, cesium, rubidium, strontium, calcium or guanidium.

[0091] In embodiments of the methods herein, the base added to quench the derivation reaction is a strong base. In embodiments, the strong base is sodium hydroxide, potassium hydroxide, barium hydroxide, cesium hydroxide, strontium hydroxide, calcium hydroxide, lithium hydroxide or rubidium hydroxide. In embodiments, the strong base is sodium hydroxide.

[0092] In embodiments of the methods herein, the base has a concentration of about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0 or 15.0 M prior to addition to the mixture. In embodiments, the base has a concentration of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0 M prior to addition to the mixture. In embodiments, the base has a concentration of from about 1.0 to about 15.0 M, from about 2.0 to about 12.0 M, from about 3.0 to about 10.0 M, from about 4.0 to about 7.0 M or from about 5.0 to about 8.0 M. [0093] In embodiments, the amount of base added is adjusted to quench the derivatization reaction as desired. In embodiments, the ratio of mixture to base in the quenched mixture is about 20:1 to about 1:5 (v/v). In embodiments, the ratio of mixture to base in the quenched mixture is about 10:1 to about 1 :5 (v/v). In embodiments, the ratio of mixture to base in the quenched mixture is about 5: 1 to about 1:5 (v/v). In embodiments, the ratio of mixture to base in the quenched mixture is about 3 : 1 to about 1 :3 (v/v). In embodiments, the ratio of mixture to base in the quenched mixture is about 2: 1 to about 1 :2 (v/v). In embodiments, the ratio of mixture to base in the quenched mixture is about 1:1 (v/v).

[0094] In embodiments of the methods herein, the addition of the base increases the pH of the quenched mixture. In embodiments, the pH of the quenched mixture is greater than about 1.0. In embodiments, the pH of the quenched mixture is greater than about 1.5. In embodiments, the pH of the quenched mixture is greater than about 2.0. In embodiments, the pH of the quenched mixture is greater than about 2.5. In embodiments, the pH of the quenched mixture is greater than about 3.0. In embodiments, the pH of the quenched mixture is greater than about 3.5. In embodiments, the pH of the quenched mixture is greater than about 4.0. In embodiments, the pH of the quenched mixture is greater than about 4.5. In embodiments, the pH of the quenched mixture is greater than about 5.0. In embodiments, the pH of the quenched mixture is greater than about 5.5. In embodiments, the pH of the quenched mixture is greater than about 6.0. In embodiments, the pH of the quenched mixture is greater than about 6.5. In embodiments, the pH of the quenched mixture is greater than about 7.0. In embodiments, the pH of the quenched mixture is about 5.5. In embodiments, the pH of the quenched mixture 5 to 10, 6 to 10, 7 to 10, 8 to 10 or 9 to 10. In some embodiments, the pH of the quenched mixture is 6 to 10, 6 to 8, or 6 to 7.5. In embodiments, the pH of the quenched mixture is about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or 7.0.

[0095] Aldehydes or ketones stabilized using the method herein may be separated and detected using any method known in the art. In embodiments, the detecting comprises liquid chromatography, mass spectrometry, or combinations thereof. In embodiments, the detecting further comprises measuring the UV absorbance of the derivatized aldehyde. In embodiments, the liquid chromatography method is a traditional gravity driven liquid chromatography method. In embodiments, the Equid chromatography is high performance Equid chromatography (HPLC). [0096] In embodiments, the mass spectrometer used in the mass spectrometry methods is a sector mass spectrometer, a time-of-flight mass spectrometer, a quadrupole mass spectrometer, an ion trap mass spectrometer or a Fourier transform ion cyclotron resonance spectrometer. In embodiments, the mass spectrometer is a tandem mass spectrometer. In embodiments, derivatized aldehyde is separated using liquid chromatography prior to analysis by mass spectrometry.

[0097] In embodiments, mass spectrometry is applied for total ion current (TIC) detecting a specific range of mass to charge (m/z) values. In some embodiments, m/z values = molecular weight (MW) of aldehyde + molecular weight of the derivatizing agent, e.g., DNPH (198 g/mol) minus H 2 O (18 g/mol) = hydrazone (molecular weight) -1 (-> negative ionization mode). In embodiments, m/z = 100-1000. In embodiments, extracted ion current (XIC) values for m/z are obtained from the TIC. In embodiments, the following m/z values are used for detection and quantification of derivatives: m/z = 335 for 4-hydroxynonenal derived with DNPH (HNE-DNPH), m/z = 319 for trans-2-nonenal derived with DNPH and m/z = 279 for hexanal derived with DNPH.

[0098] In embodiments, derivatives are detected by their absorption of UV light. In embodiments, UV light at a wavelength of 190 - 400 nm is used for detection. In embodiments, the derivatives show a UV absorbance maximum between 300 - 400 nm. In embodiments, the derivatives show a UV absorbance maximum between 320 - 380 nm. In embodiments, the derivatives show a UV absorbance maximum between 360 - 370 nm. In embodiments, the derivatives show a UV absorbance maximum at about 364 nm.

[0099] In embodiments, the present disclosure provides a method of preventing the degradation of a polysorbate to a nonenal in a sample, the method comprising adding butylated hydroxyl toluene (BHT) to the sample. In embodiments, the polysorbate is PS20 or PS80. In embodiments the nonenal is 4-hydroxynonenal.

[00100] Also disclosed are the following items:

1. A method of derivatizing a C 1 -C 20 aldehyde in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises a derivatizing agent and an acid, and ii. wherein the mixture provides a derivatization reaction between the C 1 -C 20 aldehyde and the derivatizing agent to form a derivatized aldehyde; and b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction.

2. A method of detecting a C 1 -C 20 aldehyde in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, i. wherein the derivatization solution comprises a derivatizing agent and an acid; and ii. wherein the mixture provides a derivatization reaction between the C 1 -C 20 aldehyde and the derivatizing agent to form a derivatized aldehyde; b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction, and c) detecting the derivatized aldehyde.

3. A method of detecting a C 1 -C 20 aldehyde in a sample, the method comprising: a) combining the sample with a derivatization solution to form a mixture, wherein the derivatization solution comprises an organic solvent, a derivatizing agent and an acid; ii. wherein the sample comprises an aqueous solvent; and iii. wherein the mixture provides a derivatization reaction between the C 1 -C 20 aldehyde and the derivatizing agent to form a derivatized aldehyde; b) adding a base to the mixture to form a quenched mixture, thereby quenching the derivatization reaction, and wherein the quenched mixture forms an organic phase and an aqueous phase; c) isolating the organic phase of the quenched mixture containing the derivatized aldehyde; and d) detecting the derivatized aldehyde in the organic phase.

4. The method of any preceding item, wherein the C 1 -C 20 aldehyde is an unsaturated aldehyde. 5. The method of any preceding item, wherein the C 1 -C 20 aldehyde is a mono-unsaturated aldehyde.

6. The method of any preceding item, wherein the C 1 -C 20 aldehyde is a poly-unsaturated aldehyde.

7. The method of any preceding item, wherein the C 1 -C 20 aldehyde is optionally substituted with a hydroxyl, methyl, ethyl, halogen, carbonyl, benzyl or combinations thereof.

8. The method of any preceding items, wherein the C 1 -C 20 aldehyde is optionally substituted with one or more hydroxyl substituents.

9. The method of any preceding item, wherein the C 1 -C 20 aldehyde is a C 6 -C 16 aldehyde.

10. The method of any preceding item, wherein the C 1 -C 20 aldehyde is a C 8 -C 10 aldehyde.

11. The method of any preceding item, wherein the C 1 -C 20 aldehyde is a nonenal.

12. The method of item 11, wherein the nonenal is a 2 -nonenal, a 4-nonenal or a 6-nonenal.

13. The method of any of items 1-4, wherein the C 1 -C 20 aldehyde is 4-hydroxynonenal, trans- 2-nonenal, 2-undecenal, 2-decenal, crotonaldehyde, benzaldehyde, 4-hydroxy-2-hexenal, 2-4- nonadienal or 2-4-decadienal, 4-oxononenal, formaldehyde, acetaldehyde, butanal, pentanal, hexanal, heptanal, octanal, 2-octenal, nonanal, 2 -nonenal, decanal, undecanal, dodecanal, tridecanal, acrolein, or benzaldehyde.

14. The method of any of items 1-4, wherein the C 1 -C 20 aldehyde is 4-hydroxynonenal.

15. The method of any preceding item, wherein the derivatizing agent is a substituted hydrazine.

16. The method of item 15, wherein the substituted hydrazine is an aryl hydrazine.

17. The method of item 15, wherein the substituted hydrazine is a phenyl hydrazine.

18. The method of item 17, wherein the phenyl hydrazine is substituted with one or more of nitro, hydroxyl, alkyl, halogen or combinations thereof. 19. The method of item 17, wherein the phenyl hydrazine is substituted with one or more nitro.

20. The method of item 17, wherein the phenyl hydrazine is a dihydrophenylhydrazine.

21. The method of item 15, wherein the substituted hydrazine is 2,4-dinitrophenylhydrazine, 2,5-dinitrophenylhydrazine, 2,6-dinitrophenylhydrazine, 3,4-dinitrophenylhydrazine, or 3,5- dinitrophenylhydrazine, pentafluorophenylhydrazine, 2-chlorophenylhydrazine, 2,4- dichlorophenylhydrazine, (cyclohexanedione) N-methyl-benzothiazolon-(2)-hydrazone (MBTH), 4-nitrophenylhydrazine, 1 -methyl- 1 -(2, 4-dinitrophenyl)-hydrazine (MDNPH) 1- Dimethylaminonaphthalene-5-sulfonylhydrazide (Dansyl hydrazine DNSH), Hydrazine reagent based on the benzooxadiazole backbone, 2-diphenylacetyl-l,3-indandione-l -hydrazone (DAIH) or 2,4,6-trichlorophenylhydrazine (TCPH).

22. The method of any of items 1-14, wherein the derivatizing agent is Purpald (4-Amino-3- hydarzino-5-mercapto-l,2,4-triazol, ammonia and acetylacetone or dimedone, or an O-Alkylated hydroxylamine selected from O-methylhydroxylamine, O-benzylhydroxylamine, O-(p- nitrobenzyl)hydroxylamine or O- pentafluorobenzylhydroxylamine (PFBOA).

23. The method of any preceding item, wherein the derivatization solution comprises about 0.005M to about IM derivatizing agent.

24. The method of any preceding item, wherein the derivatization solution comprises about 0.02 M to about 0.1M derivatizing agent.

25. The method of any preceding item, wherein the derivatization solution comprises about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 M derivatizing agent.

26. The method of any preceding item, wherein the mixture comprises about 0.0005M to about IM derivatizing agent.

27. The method of any preceding item, wherein the mixture comprises about 0.01 M to about 0.1M derivatizing agent. 28. The method of any preceding item, wherein the mixture comprises about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 M derivatizing agent.

29. The method of any preceding item, wherein the acid is a weak acid.

30. The method of item 29, wherein the weak acid is phosphoric acid, formic acid, acetic acid, benzoic acid, oxalic acid, hydrofluoric acid, nitrous acid or sulfiirous acid.

31. The method of item 26, wherein the weak acid is phosphoric acid.

32. The method of any of items 1, 2 and 4-31, wherein the derivatization solution further comprises an organic solvent.

33. The method of any of items 3-32, wherein a ratio of organic solvent to acid in the derivatization solution is from about 1:5 to about 5:1 (v/v).

34. The method of item 33, wherein the ratio of organic solvent to acid in the derivatization solution is about 3:1 (v/v).

35. The method of item 33, wherein the organic solvent is acetonitrile, acetone, 1,4-dioxane, tetrahydrofuran 1 -propanol, 2 -propanol, methanol, ethanol, 1 -butanol, formamide, N- methylformamide, propylenecarbonate, N,N-dimethyl formamide or N,N-dimethyl sulfoxide.

36. The method of any preceding item, wherein the sample is an aqueous sample.

37. The method of any preceding item, wherein the sample is a lab culture, food preparation, cosmetic, water sample or pharmaceutical preparation.

38. The method of item 36, wherein the sample is an aqueous sample comprising a protein.

39. The method of item 38, wherein the protein is a therapeutic protein.

40. The method of any preceding item, wherein the sample further comprises a fatty acid.

41. The method of any preceding item, wherein the sample further comprises an unsaturated fatty acid. 42. The method of any preceding item, wherein the sample further comprises an emulsifier.

43. The method of item 42, wherein the emulsifier is a polysorbate.

44. The method of item 43, wherein the polysorbate is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80.

45. The method of any preceding item, wherein the C 1 -C 20 aldehyde in the sample is leached from a packaging, a manufacturing apparatus, or an administration device.

46. The method of item 45, wherein the packaging comprises an ampule, vial, bottle, container, screw cap, carton, stopper, or tubing.

46. The method of item 45, wherein the packaging comprises a polymer, resin, metal, or glass.

47. The method of item 45, wherein the container comprises polyethylene, polyvinyl chloride, or a plastic additive.

48. The method of item 47, wherein the plastic additive in the container is epoxidized soybean oil.

49. The method of item 45, wherein the administration device is a syringe, a dropper, a spoon, or a dosing cup.

50. The method of item 45, wherein the sample is obtained by storing an aqueous solution or organic solvent in the packaging, the manufacturing apparatus, or the administration device for an extended period of time.

51. The method of item 50, wherein the extended period of time is one day to 1 year.

52. The method of any preceding item, wherein the ratio of derivatization solution to sample is from about 1:10 to about 10:1 (v/v).

53. The method of item 52, wherein the ratio of derivatization solution to sample is about 1 : 1 (v/v). 54. The method of any preceding item, wherein the pH of the mixture after the derivatization reaction is less than about 2.0.

55. The method of any preceding item, wherein the pH of the mixture after the derivatization reaction is less than about 1.0.

56. The method of any preceding item, wherein, upon dissociation, the base comprises an anion selected from fluoride, sulfate, hydrogen phosphate, acetate, chloride, nitrate, bromide, chlorate, iodide, perchlorate or thiocyanate.

57. The method of any preceding item, wherein, upon dissociation, the base comprises a cation selected from ammonium, potassium, sodium, lithium, magnesium calcium or guanidium.

58. The method of any preceding item, wherein the base is a strong base.

59. The method of item 58, wherein the strong base is sodium hydroxide, potassium hydroxide, barium hydroxide, cesium hydroxide, strontium hydroxide, calcium hydroxide, lithium hydroxide or rubidium hydroxide.

60. The method of item 59, wherein the strong base is sodium hydroxide.

61. The method any preceding item, wherein the base has a concentration of about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0 or 15.0 M prior to addition to the mixture.

62. The method of any preceding item, wherein the ratio of mixture to base in the quenched mixture is about 20:1 to about 1:5 (v/v).

63. The method of any preceding item, wherein the pH of the quenched mixture is greater than about 1.0.

64. The method of any preceding item, wherein the pH of the quenched mixture is greater than about 2.0.

65. The method of any preceding item, wherein the pH of the quenched mixture is greater than about 5.0. 66. The method of any preceding item, wherein the pH of the quenched mixture is about 5.5.

67. The method of any of items 2-66, wherein the detecting comprises liquid chromatography, mass spectrometry, or combinations thereof.

68. The method of item 67, wherein the detecting further comprises measuring the UV absorbance of the derivatized aldehyde.

[00101] All references cited herein, including patents, patent applications, papers, textbooks and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated herein by reference in their entirety.

EXAMPLES

Example 1 - 4-Hvdroxvnonenal is an Oxidative Degradation Product of Polysorbate 80

[00102] Polysorbates (PS) are used in biopharmaceutics to stabilize the therapeutic protein against interfacial stress and surface adsorption. Previous studies have shown that PS undergoes oxidative degradation. See, e.g., Kishore et al., “Degradation of Polysorbates 20 and 80: Studies on Thermal Autoxidation and Hydrolysis.” J. Pharm Set. 100(2).721-731 (2011); Zhang et al. ,"Degradation Mechanisms of Polysorbate 20 Differentiated by 18 O-labeling and Mass Spectrometry,” Pharm Res 34(l):84-100 (2017); and Donbrow et al. “Autooxidation of polysorbates,” J Pharm Sci. 67(12):1676-1681 (1978).

[00103] Based on the presence of linoleic acid at up to 18% (specified by European, Japanese and United States Pharmacopeias) in Polysorbate 80 (PS80) and the oxidative susceptibility of PUFAs leading to α,β-unsaturated aldehydic degradants, it was hypothesized that 4-hydroxynonenal (HNE) may be a PS80 oxidation product. 4-hydroxynonenal (HNE) is a peroxidation product of n-6 (poly)unsaturated fatty acids (PUFAs) e.g. linoleic acid. Due to its bi-reactive chemical structure, HNE is highly reactive towards nucleophilic amino acid of proteins, leading to protein adducts and cross-links under pathological conditions.

[00104] Since HNE could not be detected directly in the PS80 matrix by UV and mass spectrometry, a new method was developed and qualified in order to detect and quantify the potential presence of HNE in formulations containing PS80. After derivatization with 2,4- dinitrophenyl hydrazine (DNPH) and extraction of the formed hydrazone with a salting-out assisted liquid-liquid extraction, the HNE-DNPH adduct was chromatographically separated and detected by multiple reaction monitoring.

[00105] To prove that HNE is indeed a PS80 degradant, a kinetic oxidation study was conducted including 4 different stress conditions, as well as PS80 with and without the addition of the antioxidant butylated hydroxyl toluene (BHT).

[00106] With the aid of the sensitive, precise and accurate assay, HNE was indeed detected as a PS80 degradation product. The addition of BHT prevented the formation of HNE under oxidative stress conditions.

[00107] The following abbreviations are used in this Example:

ACN - Acetonitrile

API - Active pharmaceutical ingredient

AU - Arbitrary units

AAPH - 2,2'-azobis(2-amidinopropane) dihydrochloride

APCI - Atmospheric pressure chemical ionization

BHT - Butylated hydroxyl toluene

ChP - Chinese Pharmacopoeia

CE - Collision energy

DNPH - 2, 4-Dinitrophenyl hydrazine

DP - Declustering potential

EP - European Pharmacopoeia

ESI - Electrospray ionization

FAs - Fatty acids

FeSO 4 - Iron(II) sulfate H 2 O - Water H 2 O 2 - Hydrogen peroxide

HNE - 4-Hydroxynonenal

JP - Japanese Pharmacopoeia

LOD - Limit of detection LOQ - Limit of quantification

MRM - Multiple reaction monitoring NA - Not applicable

NaOH - Sodium hydroxide

NS - Not specified

POE - Polyoxyethylene

Ppb - Parts per billion is expressed in (v/v)

PS20 - Polysorbate 20

PS80 - Polysorbate 80

PUFA - Polyunsaturated fatty acid

RH - relative humidity

ROS - Reactive oxygen species RT - Ambient temperature S/N - Signal to noise ratio TIC - Total ion current

TFA - Trifluoroacetic acid

USP - United States Pharmacopoeia

XIC - Extracted ion chromatogram

%RSD - Relative standard deviation in percentage

Introduction

[00108] Polysorbate 20 (PS20) and polysorbate 80 (PS80) are nonionic surfactants that are used as excipients in therapeutic protein formulations to stabilize the active pharmaceutical ingredient (API) in a formulation, e.g. the therapeutic protein, against interfacial stresses and surface adsorption (1, 2). PS consists of three molecular entities: sorbitan, polyoxyethylene (POE) chains with different degree of ethoxylation and a mixture of fatty acids (FAs), with lauric acid and oleic acid being most prevalent in PS20 and PS80, respectively (3). The (simplified) chemical structure of PS is shown in FIG. 1A.

[00109] PS has high structural heterogeneity. The European Pharmacopoeia (EP), Japanese Pharmacopoeia (JP) and United States Pharmacopoeia (USP) all define concentration ranges for all accepted FAs including the main FA (see Table 1), based on information provided by suppliers on related to expected variability.

Table 1: FA distribution in percentage in PS20 and PS80 defined by EP, JP and USP.

FA (%) PS20 PS80

Caproic <1 ns Caprylic <10 ns Capric <10 ns Lauric 40-60 ns Myristic 14-25 <5

Palmitic 7-15 <16 Stearic <7 <6

Oleic <11 >58 Linoleic <3 <18 Linolenic ns <4 ns = not specified

[00110] Two main pathways are known for the degradation of PS: 1) the hydrolysis of the ester bond and 2) autoxidation at the POE chains and at the double bonds of unsaturated FAs. Oxidative degradation results in various degradation products, including peroxides, free FAs, degraded FA esters, and aldehydes and ketones (3, 4). It was shown that PS80 is generally more susceptible to autoxidation compared to PS20, mainly due to the higher content of polyunsaturated fatty acids (PUFAs) such as linoleic acid (chemical structure see Figure IB) and linolenic acid (5).

[00111] The degradation of PS has several effects: (a) Some of the degradation products, especially free FAs, are sparsely soluble in the aqueous formulation, potentially leading to the appearance of visible and sub-visible particles dependent on specific solubility and formulation matrix (6-11), included particles may result in an incompliant product (failing sub- visible or visible particle requirements); (b) PS degradants have been associated with adverse effects, such as adverse local tolerability for patients (12); (c) the degradation of PS also reduces the level of intact surfactant, and with this, the stabilizing effect on the active protein against interfacial stress and surface adsorption may not be maintained; and (d) other oxidative degradation products of PS, such as lauric acid, might lead indirectly to protein aggregation (10). Additionally, peroxides, increasing further in concentration during PS degradation, may interact directly with the therapeutic protein due to their oxidative properties (13). Hence, proteins may be oxidized. If oxidation happens in structurally relevant features, e.g. in the complementary determining region (CDR) of monoclonal antibodies (mAbs), the efficacy of the drug might be impaired.

[00112] In the field of lipid research, the lipid peroxidation product 4-hydroxynonenal (HNE) has gained considerable attention due to its involvement in pathogenic conditions (14-17). The chemical structure of HNE is shown in FIG. 1C. Three non-enzymatic pathways, based on the reaction of reactive oxygen species (ROS) with PUFAs, are suggested in the literature, which ultimately lead to the formation of HNE from intermediate lipid hydroperoxides, alkoxyl radicals and peroxyl radicals ( 16- 18) : a) under acidic conditions, HNE is formed from lipid hydroperoxides by Hock rearrangement followed by Hock cleavage; b) in the presence of transition metals, such as ferrous ions, HNE is produced by beta-scission of alkoxyl radicals; and c) from peroxyl radicals, HNE is released by the cleavage of the intermediately formed dioxetane. These three mechanisms are depicted in FIG 2.

[00113] HNE is bi-reactive, e.g. the α,β-unsaturated carbonyl can form Schiff bases with primary amines, and participate as Michael acceptor in electrophilic reactions. Therefore, HNE and other α,β-unsaturated aldehydes were shown to bind under physiological conditions covalently to proteins, preferably by reaction with the amino acids cysteine, histidine, and lysine, leading to protein adducts and to cross-linking of proteins, which can lead to protein aggregation and cell death (14).

[00114] Based on the occurrence of the n-6 PUPA linoleic acid in PS80 at up to 18% (see Table 1) and the known susceptibility of linoleic acid towards oxidative degradation leading to HNE formation, it was hypothesized that HNE might be an oxidative degradation product of PS80.

[00115] Previously, degradation products of PS80 with similar chemical structures, Le. α,β- unsaturated carbonyls such as 2-undecenal, 2-decenal and 2-nonenal, were reported (10), but there are no known reports of HNE as an oxidative PS80 degradation product. One purpose of this study was to investigate whether HNE is an oxidative degradant of PS80.

[00116] Since the direct quantification of HNE in the complex PS80 matrix by LC-UV-MS was not successful due to co-elution of other PS (oxidation) species with similar absorbance maximum around 220 nm for UV detection, and ion suppression for MS detection, the present disclosure provides a new assay based on 2, 4-dinitrophenyl hydrazine (DNHP) derivatization and multi reaction monitoring (MRM) LC-MS detection, to circumvent these shortcomings. After its qualification, the disclosed assay was applied on oxidatively stressed PS80 samples to demonstrate the formation of HNE as a oxidative PS80 degradation product. The disclosure further assessed if butylated hydroxyl toluene (BHT, chemical structure shown in FIG. ID), could prevent the oxidative formation of HNE from PS80 (19).

Materials & Methods

Chemicals, Reagents and Other Materials

[00117] Polysorbate 80, containing 0.2% (w/w) BHT was obtained from LONZA AG.Water (H 2 O) used during this study was purified (generated in-house, GenPure Pro UV, Thermo Scientific, Langenselbold, Germany). HNE and ethanol were obtained from Merck (Zug, Switzerland). Hexanal, trans-2-nonenal, 0.2 M 2, 4-dinitrophenyl hydrazine (DNPH) in phosphoric acid solution, heptanal-DNPH, butylated hydroxyl toluene (BHT), ammonium formate, acetic acid, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), 30% (w/w) hydrogen peroxide (H 2 O 2 ) solution, Polysorbate 80 (low peroxide) and iron(II)sulfate heptahydrate (FeSO 4 ) and needles (BD microlance, 20G, 0,9 mm x 40 mm) were obtained from Sigma Aldrich (Buchs, Switzerland). Acetonitrile (ACN) (Pierce LC-MS grade) was obtained from Thermo Scientific (Reinach, Switzerland). Dichloromethane and methanol (LC-MS grade) were obtained from Honeywell (Suhr, Switzerland). 10 M sodium hydroxide (NaOH) solution was obtained from Fisher Chemical (Reinach, Switzerland). Polysorbate 80 (J.T. Baker grade) manufactured by Croda (Reinach, Switzerland) and trifluoroacetic acid (TFA) were obtained from Thermo Fisher Scientific Schweiz AG (Reinach, Switzerland). HPLC vials (glass type 1) and screw caps containing PTFE/silicone septa were obtained from Agilent (Basel, Switzerland).

Sample preparation

[00118] Preparation of 0.05 M 2, 4-Dinitrophenyl hydrazine (DNPH) solution was as follows: 0.2 M DNPH in phosphoric acid solution was diluted with acetonitrile (ACN) to a concentration of 0.05 M DNPH (~pH 1). [00119] Sample preparations during method development were as follows:

[00120] To determine an appropriate DNPH derivatization duration, HNE, hexanal and trans-2- nonenal were separately spiked into aqueous 0.1% (w/v) PS80 (J.T. Baker grade) solutions resulting in a final concentration of 1 ppm each. Aliquots were mixed with the same volume of 0.05 M DNPH. The samples were incubated for 20 min at ambient temperature (RT) before analysis. Sample injection was performed every 1 h for ~14 h and additionally after ~48 h. Detection was performed with UV at 364 nm and with MS in negative mode extracting the ion chromatogram (XIC) at m/z = 335 for HNE-DNPH, m/z = 319 for trans-2-nonenal-DNPH, and m/z = 279 for hexanal-DNPH (see Detection by LC-UV-MS).

[00121] To determine the influence of different concentrated NaOH solutions on the stability of the HNE-DNPH hydrazone, HNE was spiked in an aqueous 0.1% (w/v) PS80 (J.T. Baker grade) solution resulting in a final concentration of 1 ppm. Aliquots were mixed with the same volume of 0.05 M DNPH solution (~pH 1). The samples were incubated for 20 min at RT before the different concentrated NaOH solutions (0 M, i.e. H 2 O, 1 M and 10 M) were added. 1 M NaOH solution was prepared from 10 M NaOH stock solution. 1 volume of NaOH solution was added to 8 volumes containing the mix of the sample and the 0.05 M DNPH solution. After vortexing, a phase separation in the samples with the added 10 M NaOH solution was observed. The samples were centrifuged for 2 min at 8 rpm to remove precipitates, and the supernatant was transferred into a new vial. Of the samples showing phase separation only the orange colored upper organic phase was transferred and analyzed. The samples were injected every 2 h for ~14 h into the LC-UV system, monitoring the UV signal at 364 nm (see Detection by LC-UV-MS).

[00122] The optimization of MRM signal intensity of the precursor/product ion transitions of 335 > 167 and 335 > 163 was performed by repeated LC-MS analysis of a HNE-DNPH standard at a concentration of 100 ppb (see 'Final standard preparation / derivatization process'). After chromatographic separation (see Detection by LC-UV-MS), variations in collision energy (CE) voltage ranging from -20 to -32 V and in declustering potential (DP) voltage ranging from -20 to -50 V were manually applied. Double injections were performed for each of the tested variations. [00123] The final standard and sample preparation / derivatization process was as follows:

[00124] To achieve an external standard line the same volume of different concentrated spiking solutions of HNE was spiked into aqueous 0.1% (w/v) PS80 (J.T. Baker grade) solution (see 'Standards used for the generation of external calibration curves'). Oxidatively stressed PS80 samples wweerree diluted to 0.1% (w/v) prior to the derivatization process. To an aliquot of each sample and standard the same volume of 0.05 M DNPH solution (~pH 1) was added and the mixture was vortexed. The samples and standards were incubated at RT for 20 min to allow the derivatization reaction to occur. To stop the reaction and to separate the organic phase from the aqueous phase 1 volume of 10 M NaOH was mixed with 8 volumes of derivatized samples and standards The exothermic reaction was controlled by storing the samples for 20 min at 2-8°C. The samples were centrifuged for 2 min at 8 rpm and the upper phase was transferred into an HPLC vial (glass type 1). If further precipitation was observed, the samples were centrifuged a second time for 2 min at 8 rpm. The precipitate-free worked-up samples and standards were transferred into HPLC vials (glass type 1) and analyzed by LC-UV-MS (see Detection by LC-UV-MS analysis).

[00125] The standards used for the generation of external calibration curves were as follows:

[00126] Six different concentrations of HNE (2 ppb, 5 ppb, 10 ppb, 20 ppb, 30 ppb, 50 ppb) were prepared in aqueous 0.1% (w/v) PS80 (J.T. Baker grade) solution and worked-up as described above (see 'Final standard and sample preparation / derivatization process'). Each standard was injected in triplicate. During the qualification of the method, lower and higher concentrations (0.5 ppb, 1 ppb and 75 ppb) were included to determine the limit of quantification (LOQ) and the linear range.

Detection by LC-UV-MS

[00127] Chromatographic separation and detection of the derivatized samples was performed on a QTRAP 6500 (Sciex, Baden, Switzerland) interfaced with a 1290 Infinite UHPLC modules including a DAD (Agilent, Basel, Switzerland) using a Luna 3u C8(2) 100 A, 50 x 2.00 mm column (Phenomenex, Basel, Switzerland) at a column temperature of 40°C. Injection volume was 20 μL. The flow was set to 0.25 mL/min. Eluent A) was 0.03% (v/v) acetic acid in H 2 O and B) was ACN. A gradient from 60% A to 35% A in 8.5 min and then in 4 min to 0% A, then for 2.5 min at 0% A, then for 2 min up to 60% A, then 3 min at 60% A. The total run time per injection was 22 min including 2 min DAD equilibration.

[00128] During development of the assay, MS analysis (ESI in negative mode) was applied in total ion current (TIC), with detection of m/z = 100-1000 in QI. From the obtained TIC the extracted ion current (XIC) at m/z = 335 for HNE-DNPH, at m/z = 319 for trans-2-nonenal-DNPH and at m/z = 279 for hexanal-DNPH was used for detection and quantification. To increase the specificity of the detection of HNE-DNPH multiple reaction monitoring (MRM) with the transition m/z 335 > 167 was implemented using a CE of -24 V and a DP of -50 V.

[00129] Additionally, UV (190 - 400 nm) was used for detection. The derivatized aldehydes showed a specific absorbance maximum at 364 nm, which was used for detection and quantification.

Qualification of the method

[00130] The qualification parameters selectivity, accuracy, precision, intermediate precision, linearity, limit of detection (LOD), limit of quantification (LOQ) and linear range were evaluated for the developed HNE-DNPH assay. HNE solutions in 0.1% (w/v) PS80 (J.T. Baker grade) were prepared at concentrations ranging from 0.5 ppb to 75 ppb. Derivatization was performed as described above (see 'Final standard and sample preparation / derivatization process'). All samples and standards were injected and analyzed in triplicate (see Detection by LC-UV-MS'). The LC- MS instrument precision was determined via the repeatability of the MRM signal intensity by injecting each prepared standard in triplicate. The work-up precision was determined by preparing three HNE standards within the determined linear range of the assay, i.e. at low, intermediate, and high concentration, in triplicate. The intermediate precision was determined by preparing three HNE standards within the determined linear range of the assay, i.e. at low, intermediate, and high concentration, on two different days by two analysts. Analyst 1 prepared the standards three times (see work-up precision) and analyst 2 prepared the standards once. Determination of unknown BHT level in low peroxide PS80 by standard addition

[00131] To determine the unknown BHT level present in the low peroxide PS80, the raw material was diluted to 0.1% (w/v) PS80 aqueous solution. The same volume of different concentrated BHT spiking solutions was added to the diluted low peroxide PS80. The resulting spiked BHT concentrations ranged from 250 to 4000 ppb. The samples were analyzed by LC-UV at an absorbance wavelength at 278 nm. The obtained calibration curve showed an off-set on the ordinate due to the presence of BHT at the unknown concentration. The unknown BHT concentration was determined by solving the linear equation. The obtained value representing the concentration in 0.1% (w/v) PS80 solution was multiplied by 1000 to achieve the initial concentration in the raw low peroxide PS80. Moreover, a dilution factor due to the addition of the spiking solutions was taken into account.

[00132] Chromatographic separation and UV detection of BHT was performed on 1290 Infinite UHPLC modules including a DAD (Agilent, Basel, Switzerland) using a Lima 3u C8(2) 100 A, 50 x 2.00 mm column of Phenomenex (Basel, Switzerland) at a column temperature of 40°C. The injection volume was 20 μL. The flow was set to 0.4 mL/min. Eluent A) was 10 mM ammonium formate and B) methanol. The gradient was for 1 min A at 95%, then to 0% A in 7 min, then for 2 min at 0% A, and then to 95% in 3 min. The total run time per injection was 15 min. UV (190 to 400 nm) was used for detection. BHT showed an absorbance maximum at 278 nm, which was used for detection and quantification.

Application of the assay/oxidative kinetic PS80 stress study

[00133] The following PS80 qualities were included in the oxidative kinetic stress study: PS80 (J.T. Baker grade) meeting EP, JP and USP specifications as a pharmaceutical excipient, PS80 provided by Lonza containing 0.2% (w/w) of the antioxidant butylhydroxytoluene (BHT), and commercially available PS80 labelled as 'low peroxide' and containing an unspecified amount of BHT (Sigma Aldrich). The unknown BHT concentration was determined to be at 0.06% (w/w) in the low peroxide PS80 raw material (see 'Determination of unknown BHT level in low peroxide PS80 by standard addition'). The three PS80 qualities were diluted with H 2 O to 10% (w/v) solutions. The resulting molar concentration of BHT in the two aqueous 10% (w/v) PS80 solutions were 0.3 mM (low peroxide) and 1 mM (Lonza). The solutions were stirred with a magnetic stirrer for at least 20 min at ambient temperature (RT) before further sample preparation steps to achieve the 4 different oxidative stress conditions were performed. An overview of all samples and conditions is provided in Table 2.

Table 2: Overview of samples and oxidative conditions for the kinetic stress study

[00134] Stock solutions containing 1.25 mg/mL FeSO 4 and 1.5% (w/v) H 2 O 2 were prepared and added to the 10% (w/v) PS80 to obtain samples containing 10 mM H 2 O 2 and 0.1 mM FeSO 4 . Additionally, TFA in a concentration of 0.5 mM was added to the samples to support the oxidative reaction by an acidic pH. Throughout the incubation the samples were kept in the dark at RT. A stock solution of 26.5 mM 2,2'-azobis(2-amidinopropane) (AAPH) was prepared and spiked to each 10% (w/v) PS80 solution to obtain samples containing 1.5 mM AAPH. Incubation was performed at 40°C / 75% relative humidity (RH).

[00135] For oxidative stress, the PS80 qualities were exposed to air at an elevated temperature of 40°C / 75% RH. Air exposure was ensured by piercing the vial stoppers with a needle. To achieve samples comparable to the other three oxidative stress conditions with regard to sample volume and PS80 dilution, respectively, H 2 O was added to each of the three aqueous 10% (w/v) PS80 solutions. Incubation was performed at 40°C / 75% RH.

[00136] The prepared sample solutions were mixed. For each kinetic time-point 1 mL was aliquoted into a 6R glass vial, closed with a vial stopper and stored at the above described storage conditions. In total four kinetic time-points were included, i.e. T1 = 1 week, T2 = 2 weeks, T3 = 3 weeks and T4 = 5 weeks of incubation. As a control, an aliquot of each diluted PS80 quality was frozen without further treatment at -20°C. The preparation of the sample solutions and the start of the incubation was performed for all conditions on the same day.

[00137] Upon reaching their time-point the samples were frozen at -20°C. As described under sample preparation, the 10% (w/v) PS80 samples were diluted with H 2 O to 0.1% (w/v) PS80 aqueous solutions before sample work-up and analysis. Each sample was injected in triplicate. The measured values were quantified by interpolation in the obtain external HNE-DNPH calibration curve, which was freshly prepared each time sample sets were derivatized. The mean concentration was calculated from the injected triplicates and multiplied by 100 to determine the HNE concentration in the incubated 10% (w/v) PS80 samples.

Results and discussion

Method development

[00138] The disclosure investigated whether HNE is an oxidative degradation product of PS80. However, a direct LC-UV-MS analysis of oxidatively stressed PS80 did not unequivocally indicate the formation of HNE. UV detection and quantification of HNE was not possible due to the co- elution of other PS species and degradants absorbing at the HNE absorption maximum at 220 nm, and MS detection was obviated by the complex PS80 matrix, consisting of numerous species that elute over a broad retention time window in reversed phase mode, leading to ion suppression (data not shown). The reason that HNE was previously not reported as a PS80 degradation product might be that by application of LC-UV-MS in screening mode this molecule is not easily detectable.

[00139] Target HNE analytics were therefore developed that are compatible with the complex PS80 matrix in which the analyte was suspected. Several groups have used DNPH to derivatize and analyze aldehydes in more or less complex matrices, e.g. food (17, 20) and tissues (18). Moreover, DNPH derivatization was used to characterize and quantify free fatty acid esters as oxidative degradation products ofPS20 (21).

[00140] The reaction mechanism of carbonyls with DNPH leading to hydrazones is shown in FIG. 3.

[00141] Hydrazones were either detected by UV absorption (17, 18, 21) or by MS-detection (17, 18, 20, 21). The method developed by Douny et al. (20) was considered, because the LC-MS multiple reaction monitoring (MRM) method was applied to quantify several aldehydes, amongst them HNE, in complex food matrices. However, direct application of the method was not possible. It became evident that LC-UV-MS analysis of aldehydic samples that were derivatized with DNPH in strongly acidic medium, e.g. as performed by Douny (21) did not work with unsaturated aldehydes, like trans-2-nonenal and HNE. This method yielded reaction products that underwent further modification (double adduct formation) within a time frame where samples within a sample set typically are stored in an autosampler during analysis (data shown in FIGS. 3 and 4).

[00142] It was determined that the reaction, after maximal hydrazone formation, had to be quenched by the addition of base. To find ideal quenching conditions, aqueous sodium hydroxide (NaOH) solutions of different concentrations, i.e. 0 M, 1 M, and 10 M, were added to the reaction mix after sufficient time for hydrazone formation. Phase separation due to a salting-out effect was observed in the sample with increasing NaOH concentration. Other salting-out effects from the addition of sodium chloride, leading to phase separation in water / ACN mixtures have been reported (Tabata et al. (22)). Without wishing to be bound by theory, it is possible that the salting- out of the organic phase was likely due to the high concentration of sodium phosphate that formed upon neutralization of the phosphoric acid with NaOH. The reaction mixtures before addition of NaOH had a pH of ~1. The pH of the sample spiked with 1 M NaOH had pH of 1.4, whereas the sample spiked with 10 M NaOH reached pH of 5.5.

[00143] To evaluate the impact of the rise in pH and the observed phase separation on the stability of the hydrazone the samples were repeatedly analyzed by monitoring the UV and MS signal FIG. 6A shows monitoring the hydrazone with LC-UV, and FIG. 4B the shows formation of the double adduct as monitored by LC-UV. [00144] Similarly to the negative control, which contained 0 M NaOH, the addition of NaOH solution 1 M did not stabilize the hydrazone, as shown by the decrease of the hydrazone peak area and the increase of the double adduct peak area. In contrast, a stable signal for the hydrazone and no signal for the double adduct was obtained for the sample treated with 10 M NaOH. These results confirmed that neutralizing the reaction mixture by addition of 10 M NaOH solutions, resulting in a phase separation, was beneficial to the stability of the hydrazone formed by reaction of DNPH with HNE.

[00145] Multiple reaction monitoring (MRM) was performed similar to the method described by Douny et al. (20). Using this method, only the instrument specific parameters collision energy (CE) and declustering potential (DP) needed to be established, which was done by repeatedly injecting a worked-up HNE-DNPH standard and thereby varying the CE and DP manually. This was necessary as ion suppression of the high salt containing reaction matrix during direct infusion was observed. We chose transitions m/z 335 > 167 (highest signal intensity) and m/z 335 > 163 (qualifier), and yielded ideal CE of -24 V and DP of -50 V. The relevant graphs are shown in FIGS. 7A and 7B.

[00146] Since the highest MRM signal was achieved using transition m/z 335 > 167 it was retained as the most important monitored and reported transition. Nevertheless, the second transition, i.e. 335 > 163, which is a known transition of DNPH derivatized carbonyls (23), was recorded as a control for all following samples (data not shown).

Method evaluation

[00147] After successful method development, the performance of the method was evaluated considering the qualification parameters selectivity, accuracy, precision, mean precision, linearity, limit of quantification (LOQ), limit of detection (LOD) and by determining the linear range.

[00148] Selectivity was shown by a more than 5 times lower MRM signal in the worked up PS80 control sample, not containing spiked HNE concentrations, than obtained in the lowest quantifiable standard at 0.5 ppb (see FIG. 8), demonstrating a negligible matrix interference and the specificity of the method. Note that the residual signal in the PS80 sample spiked with 0 ppb HNE might be due to the presence of HNE as an oxidation product. [00149] An overview of the target spiking levels and results obtained for the mean measured concentrations, the two precision experiments, i.e. instrument precision and work-up precision, and the intermediate precision can be found in Table 2.

Table 2: Overview on method evaluation results. Target spiking level, mean measured concentration, and precision and intermediate precision experiments are shown. 1) Equation of calibration curve used to calculate the concentration of the measured values of analyst 1 : y = 140348x + 147346, R 2 > 0.99, included standards (2 - 75 ppb) meeting criteria of accuracy (< 10 ppb = ± 30% and > 10 ppb = ± 20% of target spiking level); and analyst 2: y = 195392x - 624761, R 2 > 0.99, included standards (5 - 75 ppb) meeting criteria of accuracy (< 10 ppb = ± 30% and > 10 ppb = ± 20% of target spiking level)

2) Not applicable (NA) because accuracy of calculated mean concentration was not in specified range of ± 30% of the target spiking level

3) NA because samples were not worked-up in triplicate by 2 nd analyst

4) n = number of included areas per concentration

5) for information only, concentration was not included in linear range.

The LOQ was determined at 0.5 ppb, as the related reference standard showed a S/N = 150, i.e. six times higher than the control sample showing a S/N = 25, which did not contain spiked HNE. The LOD was calculated by division of the LOQ by 3.3 resulting in a LOD of 0.15 ppb. A linear range from 2 to 75 ppb showing R2 > 0.99 could be established meeting the accuracy criteria of the calculated concentrations being ± 30% of the target spiking level for concentrations < 10 ppb and ± 20% for concentrations > 10 ppb (see FIG. 9).

[00150] The obtained MRM peaks of the HNE-DNPH hydrazone showed a peak shoulder, which can probably be attributed to the less favored second stereoisomer of the derivatized unsaturated carbonyl. Since the shoulder showed in dependence of the added HNE spike concentration a correlating signal intensity, it was included in the peak integration. In summary, with the results obtained during the qualification we could show, that the HNE assay based on optimized DNPH derivatization conditions, can be applied to detect and quantify HNE in the complex PS80 matrix.

Application of the assay on the oxidatively stressed samples of the kinetic PS80 stress study

[00151] Having developed a sensitive and robust method for HNE quantification in PS matrix, it was possible to investigate whether HNE is an oxidative degradation product of PS80. To achieve this goal a kinetic study was conducted, where PS80 was subjected to oxidative stress.

[00152] In addition to the PS80 (IT. Baker grade), which met the required specifications of the EP, JP, and USP as an excipient for drug product formulations, two PS80 qualities containing the antioxidant BHT at different concentration levels were included in the study, to investigate if the presence of the antioxidant would prevent HNE formation, in accordance with the protective effect of BHT on oxidative PS degradation that we had recently reported (19). Those were PS80 provided by Lonza, containing 0.2% (w/w) of the antioxidant butylhydroxytoluene (BHT), and commercially available PS80 labelled as 'low peroxide' and containing an unspecified amount of BHT (Sigma Aldrich). The BHT concentration was determined at 0.06% (w/w) in the low peroxide PS80 raw material by standard addition approach followed by LC-UV analysis at an absorbance wavelength at 278 nm (see FIG. 10).

[00153] One stress condition was the addition of Fenton's reagent, i.e. a combination of H 2 O 2 and FeSO 4 , which is known to form hydroxy radicals that lead to hydroperoxide formation in organic compounds in the presence of oxygen (7, 9, 24). Moreover, the radical initiator AAPH was added to the PS solutions and stored at elevated temperature. The spontaneous thermal decomposition of AAPH leads to carbon centered radicals, which further reacts to peroxyl radicals in the presence of oxygen (25). The oxidant was added at 5 fold molar excess compared to lower BHT level, i.e. at 1.5 mM. Further, PS solutions were exposed to air at elevated temperature. FIG. 11 illustrates the kinetic of the oxidative stress study by monitoring HNE quantities.

[00154] During stress caused by Fenton’s reagent (FIG. 11 A) only the PS80 (J.T. Baker grade) samples not containing BHT showed an increasing concentration of HNE over the course of the incubation period. In both PS80 sample sets containing the antioxidant, HNE was not detected. Very similar results were obtained when stressing the samples at 40°C with air exposure (FIG. 11C).

[00155] The kinetics for AAPH stress are more complex (FIG. 11B). While the not stabilized PS80 (J.T. Baker grade) samples showed a maximum concentration of HNE after only one week of incubation at Tl, which decreased during the course of the study, the occurrence of HNE in the stabilized samples seemed to depend on the molar ratio of AAPH and BHT used. In the case of an excess of AAPH, as present in the low-peroxide PS80 samples with a ratio of 5, the formation of HNE was delayed compared to the not stabilized samples, as indicated by the delayed maximum HNE concentration at T3. Once the ratio was balanced, as in Lonza's PS80 samples with a ratio of 1.5, HNE was not detected as an oxidative PS80 degradation product. [00156] The most striking observation of the conducted kinetic study - confirming the initial hypothesis, was that HNE is indeed an oxidative degradation product of PS80, which can likely be attributed to the presence of n-6 PUFAs such as linoleic acid. Secondly, and in accordance to with the inventors' previous published results (19), the radical scavenger BHT was able to mitigate or at least delay the oxidative PS degradation, which is indicated by no or a delayed HNE formation, in dependence of the used stress condition and the applied ratio of AAPH to BHT.

[00157] Based on the occurrence rate of HNE in the unstabilized PS80 (J.T. Baker grade), the three applied oxidative stress conditions can be divided into two groups. Group 1) showed a steadily increasing HNE concentration over the course of the study as seen for the stresses caused by Fenton's reagent and exposure to air at elevated temperature. Whereas group 2) was characterized by the rapidly reached maximum HNE concentration that decreased over time as seen for samples stressed by AAPH at an elevated temperature.

[00158] It was previously reported that the oxidative degradation of PS follows the principle of a radical chain reaction (1, 4, 13, 26). Since the kinetics of group 1) seem to depend on the rate of initial radical formation, it can be assumed that the Fenton's reagent at the used concentration and exposure to air at elevated temperature induced oxidative PS80 degradation at the initiation phase of the radical chain reaction. This was shown by an initially slow onset of HNE formation, which can be attributed to the characteristically slow initial formation of ROS in the initiation phase, followed by the accumulation of HNE, indicating the steady degradation of n-6 PUFAs due to the increase in ROS during the propagation phase.

[00159] Therefore it can be concluded that the hydrophilic hydroxy radicals of the Fenton reagent and the ROS introduced by air exposure probably first had to be converted into lipophilic ROS in order to allow their migration into the lipophilic micelle center formed by the attached fatty acids and to enable the start of the oxidative degradation of the n-6 PUFAs. The obtained kinetics of group 2) suggests the skipping of the initiation phase of the radical chain reaction, as seen by the rapidly detected HNE maximum concentration indicating that the applied oxidative stress simulated an advanced stage of the propagation phase, characterized by a high amount of lipophilic ROS, which were immediately able to degrade the PUFAs in the micelle center. Based on these results, a conclusion could be that the use of AAPH as an oxidative stress might allow the simulation of a poor PS quality with regard to the presence of oxidizing species leading to rapid oxidative PS degradation.

[00160] The decreasing HNE concentration observed in the following weeks suggests that the equilibrium was shifted from the initial predominant HNE formation towards its degradation, suggesting that the majority of n-6 PUFAs had reacted with the ROS. Furthermore, this allows the assumption that the formed α,β-unsaturated carbonyl stayed within the micelles due to its lipophilic properties and reacted with the remaining or newly formed ROS.

[00161] In addition, the two groups can be distinguished according to their tendency to form HNE in the presence of the BHT. In group 1) both BHT-containing PS80 qualities protect the PS80 against oxidative stress, as indicated by the absence of HNE formation. While in group 2) depending on the applied ratio of AAPH and BHT the formation of HNE was observed, i.e. in the low peroxide PS80, which contained on a molar basis less antioxidative molecules than added AAPH, HNE was detected, while the PS80 of Lonza, which contained BHT in molar excess to added AAPH, seemed to be protected. The PS80 excipient already containing BHT (as supplied by Sigma) was found to not contain sufficient BHT to protect against AAPS related oxidative stress.

[00162] The inventors previously established the hypothesis that BHT as lipophilic compound probably accumulates in the lipophilic center of the micelles rather than being present in the aqueous environment (19). As it can be seen in FIG. 11 A, no HNE formation was detected for the low peroxide PS80 during the investigated period despite the addition of H 2 O 2 in a molar excess of 33 to BHT. If BHT had been present in larger quantities in the aqueous environment, the predominant hydrophilic H 2 O 2 molecules or the hydroxy radicals formed together with the ferrous ions would probably have reacted immediately with the antioxidant in an equimolar ratio. Consequently, the remaining high concentration of ROS would have led to an oxidative degradation of PS, similar to the unstabilized PS80 (J.T. Baker grade) as seen by the detection of HNE. As no HNE formation was detected, this result showed that BHT, while protecting the PS molecules from oxidative degradation, was itself protected by the PS molecules from reacting with the known oxidant. In addition, the results herein show that HNE formation was possible in this specific stabilized sample set when exposed to an excess of AAPH over BHT molecules (see FIG. 1 IB). Consequently, the disclosure further supports that the lipophilic BHT accumulated in the hydrophobic micelle center formed by the (PU)FAs and protected them from oxidative degradation depending on the applied oxidative stress and the amount of lipophilic ROS produced.

[00163] In addition to the beneficial effect of BHT observed for the stressed samples, its protection was already recognizable in the not stressed control samples (stored at - -20°C for the duration of the kinetic study), i.e. a small HNE peak was detected in the unstabilized PS80 (J.T. Baker grade) sample, whereas it was not detectable in both control samples containing BHT. Since the only preparation step of these samples was the dilution of the raw material before freezing, the detection of the small HNE peak might suggest that the not stabilized PS80 (J.T. Baker grade) raw material must already have undergone some oxidative degradation that could be likely prevented by the presence of BHT.

[00164] In summary, the obtained results of the conducted kinetic study demonstrate the applicability of our assay and confirmed the hypothesis that HNE is a PS80 degradation product. In addition, the previously reported protection of BHT against the oxidative degradation of PS (19) was further confirmed, indicating that the use of BHT as PS additive could serve as an additional safety measure by ensuring a stable product quality with regard to oxidation stability of PS, if included in sufficient quantity.

Conclusion

[00165] It is believed that there are no published data reporting that HNE is a degradation product of PS80. One of the reasons might be that the complex matrix of PS80 containing formulations obscures the direct detection of this safety-relevant molecule during screening studies. The present disclosure established and qualified an HNE quantification method, and show that HNE is indeed an oxidative degradation product of PS80. Moreover, the present disclosure shows that HNE formation could be prevented in the presence of the antioxidant BHT. HNE is a highly reactive compound towards nucleophilic residues of amino acids, forming either Michael adducts or Schiff bases resulting in covalent protein modifications (14, 17). The interaction of HNE with therapeutic proteins requires further study and it is very likely that reactions leading to protein modification might occur. Hence, the formation of HNE as an oxidative degradation product of PS80 might lead to unacceptable compromises regarding efficacy, quality and safety in drug products containing therapeutic proteins.

[00166] To a certain extent, the occurrence of HNE might be prevented by using high quality PS80 raw material containing as little ROS as possible, as an increased and faster PS degradation was observed when the PS samples were stressed by AAPH, which simulated the oxidative PS degradation at an advanced stage of the propagation phase of the radical chain reaction.

[00167] Generally, this high quality PS can be maintained by adhering to special storage conditions, i.e. at sub-ambient temperature, with nitrogen overlay, and under light protection. Another approach to ensure this high quality in terms of reducing the formation of oxidizing impurities during storage, but also due to handling errors, could be to consider adding BHT to the PS raw material before further processing steps in order to prevent autocatalytic oxidative degradation, as shown in the here reported kinetic stress study.

[00168] It should be noted that the oxidation product of BHT contains structural alerts (27) that may affect the safety and quality profile of drug products consisting of nucleophilic active pharmaceutical ingredients (API) such as present in some amino acid residues of therapeutic proteins. The potential adverse effect has already been discussed in the inventors' publications, with the conclusion that its occurrence is probably negligible (19). Another consideration to prevent or reduce HNE formation could be to rely instead on PS80 raw material containing the lowest possible amount of n-6 PUFAs, as they are likely to be the source of HNE formation. As a consequence, the formation of HNE would also be reduced to a very small and probably negligible amount.

[00169] One aim of the present disclosure was to develop an assay to enable the detection and quantification of HNE in a PS80 containing matrix. In addition to the analysis of HNE, the developed test method can also be applied to analyze other carbonyls and/or ketones. Taking into account the advantages of salt-assisted liquid-liquid extraction in bio-analytics discussed by Tang et al. (28) the assay presented here could be a valuable tool for various other applications besides the (bio)pharmaceutical industry, e.g. for the analysis of food or other bio -analytical samples. References

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9. Jones MT, Mahler HC, Yadav S, Bindra D, Corvari V, Fesinmeyer RM, et al. Considerations for the use of polysorbates in biopharmaceuticals. Pharm Res. 2018;35(8): 148.

10. Kishore RS, Kiese S, Fischer S, Pappenberger A, Grauschopf U, Mahler HC. The degradation of polysorbates 20 and 80 and its potential impact on the stability of biotherapeutics. Pharm Res. 2011;28(5):1194-210.

11. Tomlinson A, Demeule B, Lin B, Yadav S. Polysorbate 20 Degradation in Biopharmaceutical Formulations: Quantification of Free Fatty Acids, Characterization of Particulates, and Insights into the Degradation Mechanism. Mol Pharm. 2015; 12(11):3805-15.

12. Singh SK, Mahler HC, Hartman C, Stark CA. Are injection site reactions in monoclonal antibody therapies caused by polysorbate excipient degradants? J Pharm Sci.

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14. Dalleau S, Baradat M, Gueraud F, Hue L. Cell death and diseases related to oxidative stress:4-hydroxynonenal (HNE) in the balance. Cell Death & Differentiation. 2013 ;20(12): 1615- 30.

15. Gentile F, Arcaro A, Pizzimenti S, Daga M, Cetrangolo GP, Dianzani C, et al. DNA damage by lipid peroxidation products: implications in cancer, inflammation and autoimmunity. AIMS Genet. 2017;4(2):103-37.

16. Hall ED. Antioxidant therapies for acute spinal cord injury. Neurotherapeutics. 2011;8(2): 152-67. 17. Liao H, Zhu M, Chen Y. 4-Hydroxy-2-nonenal in food products: A review of the toxicity, occurrence, mitigation strategies and analysis methods. Trends in Food Science & Technology. 2019.

18. Spickett CM. The lipid peroxidation product 4-hydroxy-2-nonenal: Advances in chemistry and analysis. Redox Biol. 2013;1:145-52.

19. Schmidt A, Koulov A, Huwyler J, Mahler HC, Jahn M. Stabilizing Polysorbate 20 and 80 Against Oxidative Degradation. J Pharm Sci. 2020;109(6): 1924-32.

20. Douny C, Bayram P, Brose F, Degand G, Scippo M-L. Development of an LC-MS/MS analytical method for the simultaneous measurement of aldehydes from polyunsaturated fatty acids degradation in animal feed. Drug Testing and Analysis. 2016;8(5-6):458-64.

21. Dahotre S, Tomlinson A, Lin B, Yadav S. Novel markers to track oxidative polysorbate degradation in pharmaceutical formulations. J Pharm Biomed Anal. 2018;157:201-7.

22. Tabata M, Kumamoto M, Nishimoto J. Chemical Properties of Water-Miscible Solvents Separated by Salting-out and Their Application to Solvent Extraction. Analytical Sciences. 1994;10(3):383-8.

23. Ochs SdM, Fasciotti M, Netto ADP. Analysis of 31 Hydrazones of Carbonyl Compounds by RRLC-UV and RRLC-MS(/MS): A Comparison of Methods. Journal of Spectroscopy. 2015;2015:890836.

24. Gopalrathnam G, Sharma AN, Dodd SW, Huang L. Impact of stainless steel exposure on the oxidation of polysorbate 80 in histidine placebo and active monoclonal antibody formulation. PDA J Pharm Sci Technol. 2018;72(2): 163-75.

25. Niki E. [3] Free radical initiators as source of water- or lipid-soluble peroxyl radicals. Methods in Enzymology. 186: Academic Press; 1990. p. 100-8.

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Example 2 - Determination of 4-Hvdroxvnonenal: A novel leachable of administration materials used in clinical nractice

Introduction

[00170] The migration of chemicals from processing materials into (bio)pharmaceutical products can lead to various problems, e.g. being toxic themselves or reacting with formulation components including the active pharmaceutical ingredient. Therapeutic proteins, which generally are susceptible to chemical modifications, could be especially affected. Materials used in the processing steps closer to the patient, i.e. the drug product administration, pose a particularly high risk.

[00171] Aim: The aim of this study was to test the hypothesis that the a,p-unsaturated aldehyde 4-hydroxynonenal (HNE), capable of forming Schiff bases and Michael adducts with susceptible amino acid residues leading to protein modifications and cross-links, may leach out of clinical administration kits into the drug product solution.

[00172] Methods: To test the working hypothesis, extracts of the three administration sets were analyzed using a HNE-specific assay that has recently been established, allowing the identification of the α,β-unsaturated aldehyde in complex matrices based on its derivatization with 2,4- dinitrophenylhydrazine (DNPH), liquid extraction of the formed hydrazone, followed by LC- MRM analysis.

[00173] Results: HNE was confirmed as a detected leachable of the administration sets. Moreover, the individual primary HNE-releasing materials of the disassembled sets were identified.

[00174] Conclusion: Using a sensitive HNE-DNPH assay enabled the confirmation of the highly toxic HNE as a leachable of PVC administration materials used in clinical practice. Its confirmed leaching propensity suggests an increased risk to patients, not only because of its known toxicity, but also because of potentially resulting protein modifications, impairing the quality and safe administration of susceptible drug products. Abbreviations

ACN - Acetonitrile

APCI - Atmospheric pressure chemical ionization

API - Active pharmaceutical ingredient

BHT - Butylated hydroxyl toluene

BPOG - BioPhorum Operations Group

DAD - Diode-array detector

DCM - Dichloromethane

DEHP - Di(2-ethylhexyl) phthalate

DNPH - 2,4-dinitrophenylhydrazine

DP - Drug product

ESBO - Epoxidized soybean oil

ESI - Electrospray ionization

GC-MS - Gas chromatography coupled to mass spectrometer

HNE - 4-hydroxynonenal

HS-GC-MS - Headspace gas chromatography coupled to mass spectrometer

LC-UV-MS - Liquid chromatography coupled to ultraviolet and mass spectrometer detectors m/z - mass to charge ratio

MRM - Multiple reaction monitoring

PA - Polyamide

PES - Polyethersulfone

PP - Polypropylene

PS - Polysorbate or Tween

PTFE - Polytetrafluoroethylene

PUFA - polyunsaturated fatty acid

PUR - Polyurethane

PVC - Polyvinylchloride

RT - Ambient temperature

TIC - Total ion current

XIC - Extracted ion chromatogram Introduction

[00175] During the different processing steps of biopharmaceuticals, the formulation containing the therapeutic protein (active pharmaceutical ingredient, API) is exposed to with a wide variety of (plastic) materials.

[00176] It is known that the matrix formed by the different formulation ingredients might enhance the migration of chemical compounds from the contact-materials into the aqueous drug product (DP) formulation (1). The formulation properties supporting the migration can mainly be attributed to surface-active, amphiphilic components such as the API itself, i.e. the therapeutic protein, and some protein-stabilizing excipients, such as the non-ionic surfactants polysorbate (PS) 20 and 80. The surfactants are normally added in concentrations above their critical micelle-forming concentration (CMC) to protect the proteins against interfacial stress and surfrice adsorption by competitive displacement reactions due to the excess of surfactant molecules over protein molecules (2). Moreover, the formed PS micelles may solubilize compounds, which are normally poorly soluble in an aqueous environment, including for example free fatty acids (3-7) being known degradation products of PS (8-10).

[00177] An additional advantage of PSs can therefore be seen in their ability to prevent to a certain extent the formation of visible and sub- visible particles in the final DP.

[00178] On the other hand, the solubilizing power of the surfactants lead to an equilibrium shift and an increased tendency of less polar chemical compounds to migrate from the processing materials into the aqueous formulation. The occurrence of such chemical compounds could negatively impact the quality and safety of the DP.

[00179] All materials that come into contact with the DP formulation during the various biopharmaceutical processing steps must be examined for potentially released chemicals. Depending on the extraction conditions, the migrating chemical compounds are classified as "leachables" or "extractables".

[00180] Compounds that migrate under relevant pharmaceutical conditions regarding parameters such as extraction solvent, temperature, pH and incubation time are called "leachables". In contrast, "extractables" are chemical substances that are extracted from materials under accelerated laboratory conditions such as elevated temperatures, use of strong solvents and exposure to an increased surface area that are not necessarily relevant to pharmaceutical practice. Leachables are typically a subset of extractabtes (1, 11). Therefore, extractable studies are typically conducted prior to teachable studies to allow for later target analysis of potentially migrating chemical compounds. It needs to be mentioned that the obtained extractabtes profile leaves an uncertainty regarding the actually occurring leachables, since not all detected compounds might be migrating under the relevant conditions and moreover other compounds might appear.

[00181] The BioPhorum Operations Group's (BPOG) "Best Practices Guide for Evaluating Leachables Risk in Biopharmaceutical Single-Use Systems" describes the performance of an extractable and teachable assessment as a risk-based approach, which should take into account but is not limited to (a) the exposure temperature, (b) the exposure duration, (c) the process fluid interaction with regards to solvation power and polymeric penetration, (d) the dilution ratio, which is defined by the volume and the contact area, and (e) the distance along the production stream. In general, the later in the biopharmaceutical processing a compound is teaching, the lower the probability that it will be captured in a purification step, which goes along with an increasing risk for the patient. In this respect, one of the greatest risks is posed by materials used in the DP administration process.

[00182] In general, the extracts obtained from materials incubated under carefully chosen conditions are analyzed with different approaches to ensure the detection of the majority of migrated compounds. Analysis by liquid chromatography coupled to ultraviolet detector and mass spectrometer (LC-UV-MS) is often performed to detect non-volatile compounds, gas chromatography coupled to mass spectrometer (GC-MS) to detect semi-volatile compounds and headspace gas chromatography coupled to mass spectrometer (HS-GC-MS) to detect volatile compounds.

[00183] During the course of a simulated administration leachable study, the obtained UV chromatograms of the extracts of three administration sets showed a similarly eluting peak, i.e. a leachable, at an absorption wavelength at 220 nm. [00184] The high-resolution mass spectra underlying the detected UV peaks showed a signal with a mass to charge ratio (m/z) at 157, resulting in a proposed molecular formula of C9H17O 2 for the unknown compound.

[00185] Based on these results, we hypothesized that the unknown leachable could be a highly reactive α,β-unsaturated aldehyde, namely 4-hydroxynonenal (HNE, Formula 1).

Formula 1 : Chemical structure. 4-hydroxynonenal (HNE)

[00186] HNE became known as a highly reactive peroxidation product of phospholipids containing n-6 polyunsaturated fatty acids (PUFAs), such as linoleic acid.

[00187] Its formation was previously extensively studied with regards to pathological events (12- 15) and food products (16-18). The exceptional reactivity and cytotoxicity of the α,β-unsaturated aldehyde can be attributed to its electrophilic bi-reactive chemical structure, which allows its simultaneous participation in Michael reactions as well as in Schiff base formations e.g. with nucleophilic amino acid residues of proteins (12). In addition to the formation of simple covalent protein modifications, HNE can therefore act as a cross-linking agent of proteins.

[00188] Since the high reactivity of HNE is not related to the endogenous milieu, the confirmation of its suspected occurrence as a leachable could go along with increased safety risk for patients, which is related to the potential modification of therapeutic proteins (and other nucleophilic APIs) by HNE during the administration process, as well as to its direct toxicity.

Materials and Methods

Chemicals, Reagents and other Materials:

[00189] HNE was purchased from Merck (Zug, Switzerland). 0.2 M 2,4-dinitrophenylhydrazine (DNPH) in phosphoric acid solution and polysorbate 80 (low peroxide) were purchased from Sigma Aldrich (Buchs, Switzerland). Polysorbate 20 (J.T. Baker grade) and polysorbate 80 (J.T. Baker grade) manufactured by Croda (Reinach, Switzerland) were purchased from Thermo Fisher Scientific Schweiz AG (Reinach, Switzerland). All glass syringe (50 mL, Fortuna Optima) and Hamilton syringes (1000 μL) were purchased from VWR (Dietikon, Switzerland). All other chemicals were of analytical and solvents were of chromatographic grade.

Methods:

Simulated administration leachable study

[00190] Three administration sets (A-C) that are used in clinical practice were in scope of the simulated administration leachable study and are schematically illustrated in Figure 12.

[00191] Administration set A and B had as primary containers IV bags (#1 and #6, respectively), and set C a disposable syringe (#7). Table 3 provides further details of the included individual administration materials such as their assigned material numbers (#).

Experimental set-up administration set A & B:

[00192] The IV bags (100 mL, #1 and #6) containing 0.9% (w/v) NaCl were emptied and refilled completely with a drug product (DP) surrogate solution, i.e. 0.1% (w/v) polysorbate 20 (PS20), using an all-glass syringe and were incubated for 15 h at ambient temperature (RT) exposed to ambient light. After the incubation of the two IV bags, the two administration sets (A and B) were assembled including the individual materials described in Table 3 and illustrated in Figure 12.

[00193] Table 3: Overview on the three investigated administration sets. Packaging, including individual administration materials, assigned material numbers and the polymeric composition of the material that comes into contact with the drug product surrogate formulation are shown.

[00194] The prepared administration sets were connected to a peristaltic pump and a flow of 16.6 mL/h was applied such that the bags were emptied within 6 h, i.e. the contact time of the surrogate solution with all involved materials was as well 6 h. The simulated administration was executed at

RT under ambient light. The resulting extracts were collected in glass bottles, closed and frozen in an upright position at -20°C prior to further analysis.

Experimental set-up administration set C:

[00195] The disposable administration syringe (50 mL, #7) was completely filled with a drug product surrogate solution, i.e. 0.1% (w/v) PS20, and incubated for 2 h in an upright position at RT and ambient light. After the incubation, the administration set C was assembled including the individual materials described in Table 3 and illustrated in Figure 12. The prepared administration set was connected to a peristaltic pump and a flow of 16.7 mL/h was applied such that the disposable syringe was emptied within 3 h, i.e. the contact time of the surrogate solution and all involved materials was as well 3 h. The simulated administration was executed at RT under ambient light. The resulting extract was collected in a glass bottle, closed and frozen in an upright position at -20°C prior to further analysis.

LC-UV-MS analysis of administration set extracts

[00196] For the analysis by LC-UV-MS, the three extracts of the simulated administration leachable study were thawed and subjected to liquid- liquid extraction. As a negative control 0.1% (w/v) aqueous PS20 solution was worked-up simultaneously. 10 g of each sample solution were weighted in a glass vial (20 mL). Afterwards 0.3 g of solid NaCl and 0.1 mL of a 10% (w/v) aqueous PS20 solution were added. The prepared samples were extracted three times by 1 mL dichloromethane (DCM) introduced with Hamilton syringes (1000 μL). The aqueous and organic phases were mixed using glass Pasteur pipettes. The samples were centrifuged for 10 min at 3000 G and the lower DCM phase was withdrawn using Hamilton syringes (1000 μL).

[00197] The three collected DCM volumes per sample were merged and the solvent was evaporated overnight. The samples were dissolved in 0.5 mL of a mixture of 95 volume parts of 10 mM ammonium formate and 5 volume parts of MeOH using a Hamilton syringe and analyzed.

[00198] Chromatographic separation and detection of the extracted and concentrated leachables was performed on an Orbitrap Fusion Lumos MS detector (Thermo Fisher, Basel, Switzerland), interfaced with UHPLC Vanquish liquid chromatography system including a diode-array detector (DAD) module (Thermo Fisher, Basel, Switzerland) using a Luna 3u C8(2) 100 A, 50 x 2.00 mm column (Phenomenex, Basel, Switzerland). The column temperature was set to 40°C. Injection volume was 25 μL. The flow was set to 0.4 mL/min. Eluent A was 10 mM ammonium formate in H 2 O and B - MeOH. The following gradient was used: for 1 min 5% B, then in 7 min to 100% B, then for 4 min at 100% B. The total cycle time per injection was 12 min.

[00199] Mass analysis (atmospheric pressure chemical ionization (APCI) in positive mode) was applied in total ion current (TIC), with detection of m/z = 100 - 2000. In addition, UV (190 - 400 nm) was used for detection. The resulting chromatograms were analyzed at an absorption wavelength at 220 nm.

Confirmation of HNE as the unknown administration set teachable

[00200] To confirm the presence of HNE as a leachable of the three administration sets, the obtained extracts of the simulated administration leachable study were thawed. HNE reference standards ranging from 2 to 50 ppb were prepared in 0.1% (w/v) PS80. Commercially available low peroxide PS80 (Sigma Aldrich) containing the antioxidant butylated hydroxy toluene (BHT) was used as a negative control. [00201] To an aliquot ofthe samples the same volume of the 0.05 M DNPH derivatization reagent (~pH 1, 0.2 M DNPH in phosphoric acid solution diluted by acetonitrile (ACN)) was added followed by an incubation period of 20 min at RT. One volume of 10 M NaOH was mixed with eight volumes of the derivatized samples leading to an exothermic neutralization reaction, which was controlled by storing the samples for 20 min at 2-8°C, and phase separation. The samples were centrifuged for 2 min at 8 rpm. The precipitate-free upper organic phase was transferred and analyzed.

[00202] Chromatographic separation and detection of the formed HNE-DNPH hydrazones was performed on a QTRAP 6500 (Sciex, Baden, Switzerland) interfaced with 1290 Infinite UHPLC modules (Agilent, Basel, Switzerland) using a Luna 3u C8(2) 100 A, 50 x 2.00 mm column (Phenomenex, Basel, Switzerland) at a column temperature of 40°C. Injection volume was 20 μL. The flow was set to 0.25 mL/min. Eluent A) was 0.03% (v/v) acetic acid in H 2 O and B) was ACN. A gradient from 60% A to 35% A in 8.5 min and then in 4 min to 0% A, then for 2.5 min at 0% A, then for 2 min up to 60% A, then 3 min at 60% A was used. The total run time per injection was 22 min including 2 min DAD equilibration. Mass analysis (electrospray ionization (ESI) in negative mode) was applied using a multiple reaction monitoring (MRM) approach monitoring the HNE-DNPH hydrazone specific precursor/product ion transitions 335 > 167 and 335 > 163 using a collision energy of -24 V and a declustering potential of -50 V. Since similar results were obtained for the two transitions, only data obtained from the main transition, i.e. 335 > 167, showing a higher signal intensity were reported in the course of this manuscript.

Screening experiment determining individual HNE-releasing materials

[00203] The 0.9% (w/v) NaCl containing IV bags were emptied with an all-glass syringe. An aliquot of each saline solution was analyzed without further preparation. To the emptied IV bags and other materials ofthe three administration sets 1 mL of Methanol (MeOH) was introduced by Hamilton syringes. The contact of the solvent to all infusion-relevant surfrices ofthe materials was ensured. The contact time of the organic solvent with each material was below 10 min. As reference standards, HNE was spiked in concentrations ranging from 10 to 100 ppb in MeOH.

[00204] Chromatographic separation and detection was performed on 1290 Infinite UHPLC modules including a DAD (Agilent, Basel, Switzerland) using a Luna 3u C8(2) 100 A, 50 x 2.00 mm column (Phenomenex, Basel, Switzerland) at a column temperature of 40°C. Injection volume was 25 μL. The flow was set to 0.4 mL/min. Eluent A) was 10 mM ammonium formate in H 2 O and B) was MeOH. The following gradient was used: for 1 min 5% B, then in 7 min to 100% B, then for 4 min at 100% B. The total cycle time per injection was 12 min. UV (190 - 400 nm) was used for detection. The resulting chromatograms were analyzed at an absorption wavelength at 220 nm.

Confirmation of suspected HNE-releasine materials

[00205] To the three suspected HNE-releasing administration materials, i.e. emptied IV bag (#1) and both administration lines (#2 and #8), 2 mL of 0.1% (w/v) PS20 were added and the contact of the solvent to all infusion-relevant surfaces of the materials was ensured. As reference standards, HNE was spiked in concentrations of 30 and 100 ppb in 0.1% (w/v) PS20. As a negative control, ACN was added in the same volume ratio to 0.1% (w/v) PS20 as present in the positive controls containing spiked HNE. The obtained extracts and controls were subjected to the DNPH- derivatization procedure and LC-MRM analysis as described above.

Results and Discussion

[00206] A simulated administration leachable study was conducted to detect and identify potentially occurring chemicals migrating during the infusion process from the administration materials into the drug product (DP). The study examined three administration sets that consisted of materials commonly used in clinical practice for the administration of biopharmaceutical drug products to patients. The simulation of the administration process was performed under real-use conditions using the assembled administration materials and 0.1% (w/v) PS20 as a DP surrogate solution. Table 3 provides a detailed overview of the administration sets (A-C) including the individual materials used, the assigned individual material numbers (#) and the polymeric composition of the materials the surrogate solution gets in contact with during the administration process. The schematic assembly of the three administration sets is shown in Figure 12.

[00207] The contact time of the surrogate solution and the assembled materials of administration sets A and B was 6 h and of administration set C 3 h, which represents worst-case hold-times that may also occur in clinical practice. [00208] The obtained administration set extracts were concentrated and analyzed by LC-UV-MS using a high-resolution MS to enable structure elucidation of potentially detected leachables (for details, see Materials and Methods). Beside other signals, an absorbance peak at wavelength 220 nm was detected in the resulting LC-UV-MS data, eluting after 6.6 min in all three concentrated extracts, but not detectable in the negative control. The LC-UV-MS results of the extract of administration set A with regards to the detected unknown leachable are shown in Figure 13.

[00209] Results of administration set A are shown in Figure 13. (A) UV chromatogram at an absorbance wavelength at 220 nm showing a peak, i.e. a leachable (black framed), eluting after 6.6 min. (B) Extracted ion chromatogram (XIC) at m/z = 157 showing a peak correlating well with the detected UV peak. (C) The corresponding high-resolution mass spectrum of the detected leachable showing a prominent signal at m/z = 157 (A 2 ppm) with a proposed molecular formula ofC9H17O 2 .

[00210] The corresponding high-resolution mass spectrum of the detected UV peaks at an absorbance wavelength at 220 nm eluting after 6.6 min (see Figure 13 A) showed that the unknown compound had a prominent signal at m/z = 157 with a possible molecular formula of C9H17O 2 (see Figure 13C). The resulting extracted ion chromatograms (XIC) at m/z = 157 showed a good correlation to the detected UV peak (see Figure 13B). Based on these results and the suggested molecular formula, it was hypothesized that the detected unknown leachable might be the highly reactive a,|3-unsaturated aldehyde 4-hydroxynonenal (HNE).

[00211] To confirm that HNE was the unknown leaching compound, we analyzed the extracts of the three administration sets with an assay developed to enable specifically the detection and precise quantification of HNE in a complex matrix. The HNE-DNPH assay is based on the derivatization of the aldehydic moiety of HNE with 2,4-dinitrophenylhydrazine (DNPH) at a very acidic pH, resulting in the formation of a hydrazone. To mitigate follow-up reactions due to the bi-reactivity of HNE, the samples were neutralized and the formed hydrazone was extracted from the aqueous derivatization matrix by salting-out assisted liquid-liquid extraction. The resulting upper organic phase containing the stable HNE-DNPH-hydrazone was analyzed by LC-MS using a multiple-reaction-monitoring (MRM) approach monitoring its specific precursor/product ion transition at m/z 335 > 167. HNE reference standards were prepared as positive controls. We previously showed that HNE was an oxidative degradation product of PS80 if not properly stabilized by the antioxidant butylated hydroxy toluene (BHT). Therefore, we used as a negative control commercially available low peroxide PS80 (Sigma) containing BHT.

[00212] In Figure 14 the overlay of the obtained LC-MRM signals of the HNE-DNPH hydrazone in the administration set extracts and controls using the transition 335 > 167 of the worked-up extracts of administration set A, B and C, a positive control, i.e. HNE reference standard at 30 ppb, and a negative control not containing HNE. Triplicate injections are shown.

[00213] As it can be seen in Figure 14, HNE was confirmed to be present in the extracts of all investigated administration sets. The total leaching amount per administration set was calculated by interpolation of the average MRM area of the injected triplicates into the linear calibration curve (R 2 > 0.99) obtained from the worked-up HNE standards. The resulting concentration (ppb, i.e. μg/L) was multiplied by the total extraction volume, i.e. for administration set A and B by 0.1 L and for set C by 0.05 L. The total amount of HNE leaching from administration set B was 4.6 μg and from set C 1.9 μg, both with relative standard deviations below 2%. Whereas HNE was detected also as a leachable from administration set A, its total amount in this sample could not be determined accurately, since the obtained average MRM area was below the reporting limit of the calibration curve. Since we have identified HNE as an oxidative degradation product of PS80, a valid objection could be that HNE might not actually leach from the administration materials, but is rather formed by the oxidative reaction of PS20 with another unknown oxidizing leaching chemical

[00214] In order to identify the individual materials of the administration sets as primary HNE- releasing sources and to clarify the objection made, the nine individual materials were extracted separately with a small volume of MeOH. In addition, aliquots of the removed saline solutions of the two IV bags were examined. As positive controls, HNE was spiked in different concentrations in MeOH. The samples and controls were analyzed by LC-UV at a wavelength at 220 nm, i.e. the absorbance maximum of HNE. Figure 15 shows the obtained HNE areas of the samples that corresponded to the UV signals of the reference standards.

[00215] In Figure 15, UV signals obtained in the MeOH extracts of the individual administration materials at an absorbance wavelength at 220 nm corresponding to reference standards are shown, i.e. only materials #1, 2 and 8 showed a HNE-positive response. The columns are labeled according to the related material numbers assigned in Table 3. The results of the additionally examined saline solutions of the IV bags are labeled with 1* and 6*, respectively. Values are means ± SD, n=3.

[00216] As it can be seen in Figure 15, only three of the nine materials examined showed a positive UV response for HNE that was consistent with the reference standards, namely one of the two IV bags (#1) and both administration lines (#2 and #8). Furthermore, these results verified that HNE was a genuine leachable and not an oxidative degradation product of PS20.

[00217] To confirm the three identified materials as primary HNE sources the more selective HNE-DNPH assay (derivatization of the α,β-unsaturated aldehyde by DNPH and detection of the formed hydrazone by LC-MRM) was employed. The three suspected materials were separately extracted with a small volume of 0.1% (w/v) PS20. HNE reference standards were prepared as positive controls. As negative control 0.1% (w/v) PS20 not containing HNE was used. The obtained extracts and controls were worked-up and analyzed using the HNE-DNPH assay, as described above.

[00218] In Figure 16 the overlay of the obtained LC-MRM signals of the formed HNE-DNPH hydrazone in the samples and controls is shown. Figure 16 shows overlay of obtained HNE-DNPH hydrazone MRM signals using the transition 335 > 167 in the derivatized extracts of the three suspected materials, i.e. the IV bag (#1) and the two administration lines (# 2 and #8), a positive control, i.e. HNE reference standard at 100 ppb, and a negative control not containing HNE. Triplicate injections are shown.

[00219] As it can be seen in Figure 16, the overlay of the obtained HNE-DNPH hydrazone MRM signals confirmed the presence of the derivatized HNE in the extracts of the three suspected administration materials. The obtained leaching propensities were similar to those obtained from the MeOH extracts of the previously conducted material screening study, i.e. the two administration lines showed a higher release of HNE compared to the IV bag. However, it should be noted that HNE was already detected in the saline solution of the IV bag during the previously described material screening study, indicating that a portion of HNE had already been removed prior to extraction by 0.1% (w/v) PS20. To summarize, the performed experiment applying HNE- DNPH assay led to the confirmation of the three suspected materials as primary HNE-releasing sources.

[00220] As a follow-up, the three confirmed and six remaining administration materials were compared in terms of their composition (see Table 3) to further assess the occurrence of HNE. According to the information provided the manufacturers, it was found that all primary sources of HNE were made of PVC, while all other materials were made of a different polymers.

[00221] This correlation led us to hypothesize that the occurrence of HNE as leachable in the administration set extracts might be attributed to the contact of the surrogate solution with PVC- containing material during the simulated infusion process.

[00222] Two groups of additives are commonly used in the PVC manufacturing process to achieve polymeric materials containing certain properties in terms of flexibility and resistance to heat and light, namely plasticizers and co-stabilizers. Epoxidized vegetable oils, such as epoxidized soybean oil (ESBO) or linseed oil, are combining to a certain extent the features of both additive groups (19), i.e. they increase the flexibility of the polymeric chains and stabilize the polymer by scavenging its degradation product hydrochloric acid, which is released under the influence of heat and light. Epoxidized vegetable oils consists to a greater portion of PUFAs (19, 20). Especially n-6 PUFAs, such as linoleic acid being present in ESBO in concentrations up to 57% (19), are known to form HNE during their oxidative degradation (15, 18, 21).

[00223] Therefore, one plausible hypothesis is that the occurrence of HNE as a leachable is due to the degradation of n-6 PUFAs contained in these co-stabilizers.

[00224] To our knowledge the interaction of HNE and therapeutic proteins was not yet studied. Nevertheless, their modification by HNE is likely as it was observed for endogenous proteins during physiological and pathological events (12, 15, 22). Hence, the confirmed leaching of HNE from the administration materials suggests a two-fold increased risk for patients, firstly due to its known toxicity and secondly by potentially occurring API modifications impairing a safe administration of drug products.

Conclusion [00225] The highly reactive α,β-unsaturated aldehyde HNE was not previously reported as a leachable of drug product clinical administration materials. A recently developed selective HNE- DNPH assay allowed confirmation of the suspected highly reactive chemical as a detected unknown leachable in the extracts of three administration sets used in clinical practice. As primary HNE-releasing materials both administration lines as well as one of the two investigated IV bags could be identified. Interestingly, all determined primary sources consisted of PVC, while all other materials that did not release the reactive chemical were based on a different polymer. Hence, it is likely that the occurrence of HNE as a leachable in the administration set extracts can be attributed to the exposure of PVC material during the simulated infusion process.

[00226] As HNE itself is a toxic compound, known to modify and cross-link proteins, its leaching rate from PVC administration materials should be properly understood, monitored and reduced to a minimum in order to limit the introduction of HNE to DP and patients.

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