A MET BOB FOR DEVELOPMENT OF RECOMBINANT PROTEINS WITH

Pi GERP T LIKE SIMILARITY TO THE REFERENCE PRODUCT

FIELD OF THE INVENTION

[0001 ] The present invention relates to the methods of developing recombinant proteins with a fingerprint like similarity to the reference product or the originator. The method is particularly useful, in the development of hioaimilar products. This method can also be used to establish comparability during the manufacturing process change for the originator products,

[0002] T e methods described herein are used to obtain a recipe for the production of a biosiroilar product, or a recombinant protein using a process that may be different from the original but that yields, a recombinant protein that has fingerprint level of similarity to she reference product. The methods described herein can also used to obtain a fingerprinting analysis package for a. biosimilar that can be submitted to a regulatory agenc for abbreviated biosimilar approval,

BACKGROU D OF THE INVENTION ί 0003] Recombinant proteins are a major class of biologic drugs used to treat a wide range of diseases. They are called biologies as they are produced in living cells. Production of .recombinant proteins in cells is complicated by the tact that a cell's host proteins can modify recombinant proteins by adding a variety of modifications to the product and making a product heterogeneous. This heterogeneity results in a recombinant protein, product thai is a complex mix: are of different recombinant protein product variants, each variant characterized by having a different combination of modifications.

[0004] Biosirni!ars are copies of the originator recombinant proteins. They are called bio-similar and not bio-generic as they are not identical to the originator; the term "generic ⁵ implies structural identity. Biosimisars with a fingerprint level of similarity are copies of the originator recombinant proteins that are almost indistinguishable from the originator on the analytical level and in some eases could be classified as bio-generic, or bio-identical.

[0005] A major reason for producing a recombinant protein with a fingerprint like similarity s to:

a- ensure same product safety and efficacy as the original product, the

originator,

b. limit development cost to obtain market approval for a biosiroiiar product

[0006] Thus far, producing indistinguishable biosiniilar or a bio-generic has not been possible.

[0007] The methods described herein delineate how to produce recombinant proteins with a fingerprint level similarity to the reference product and how to produce biosimiiars with a fingerprin similarity to products from third parties, such as originator products.

[0008] The methods described herein delineate the analytical methods for showing fingerprint level similarity of the biosimilar to a third party's product.

[0009] While the idea of fingerprinting has been described in Kozlowski et esL,

20.1 1 , indicating that, a rigorous "fingerprint" similarity could remove many of the uncertainties of the hiosirnilar product relative to the originator, thus far a method for fingerprinting" has yet to be developed. The challenge with developing such a methodology is that biologies are complex mixtures of many product variants, where each variant may have -a combination of different modifications. For example, different manufactured antibody lots produced even by the same company could have different modifications including but not limited to giycans, oxidized ammo acids, aggregated forms, and C-terminal lysines, When all of these modifications are taken into account, there is the potential for tens of thousands of product variants within each lot, each with the possibility to influence biological activity to different degrees.

[0010] For purposes of this specification it is important to understand the difference between a product variant and a product modification that exists on a protein. While currently available analytical methods such as mass spectrometry, chromatography and others can identity and qnantiiate specific mod fications on a recombinant protein, no methods currently exist to measure and determine the concentration of product variants in a complex -mixture. Each product variant is composed of the recombinant protein with a specific subset of modifications and complex biologic mixtures are composed of many product variants.

[001 1 ] Product modifications include but are not limited to glyeosylaiion, earboxylation, deanvklation, oxidation, hydroxylatkm, O-sulfafion, amidation, glycylation, glycation, alkyiatlon, acylation, acetylation, phosphorylation, bfotinylation, fonny!aiion, iipidation, iodination, preny!ation, oxidation, palraitoyiation, phosphatidylinositolation, phosphopantetheinyiation, sia!ylation, and se!enoytation, C terminal Lysine removal. 00! 2] The analytical methods applicable to the present disclosure mc!ude those that are capable of identifying and/or quantitatmg the modifications present on

recombinant proteins and then identifying and quantitating product variants in a complex mixture, some of which may utilize various in siiico computational approaches using the analytical data as input to derive a product variant distribution.

[00131 The in siiico computational approaches that may be used to identi y product variants from the analytical data identifying and quantitating product modification data include but are not limited to simulation, neural networks and artificial intelligence,

[00 Ί 4] To develop a biosimilar recombinant protein with a fingerprint level similarity, the distribution of product variants in biosimilar product lots must fit within the range of the distribution observed for all tested originator or reference product lots, which are likely to have slightly different product variant distributions.

[0015] If small differences in product variants are present in a biosimilar product a compared to the originator, these product variants can be assessed for their biological activit using the fingerprinting platform described herein via. structure-activity- relationship (SAR). While SAR i routinely established for small molecules, such methodology has not yet been developed for biologic products. In essence for a recombinant protein the SAR is defined by the relationship between a modification and its effect on biologic activity. [0016] The computations! approaches that may be used, to establish SAR equation include but are not limited to neural networks, multivariate anal sis, Partial Least

Squares Regression (PLS ), Principal Components Regression (PCR), artificial intelligence and machine learning.

[00 I 7] To establish SAR for a said recombinant protein one has to understand the impact of the various modifications alone and In combination on th biological activity of said recombinant protein, in order to achieve this level of understanding, one has to produce the recombinant protein enriched for each modification and. test, those variants in biological assay to determine the impact, it is expected that; a. some modifications will have no effect on biological activity,

h. other modifications will have a profound effect on biological activity, c. it is also anticipated that combinations of some modifications may have synergistic or additive effects on biological activity.

[0 181 SAR is used to determine whether specific product variants may negatively or positively impact biological activity. These variants can then be varied in concentration or eliminated by changing production processes.

[0019] There are two ways to change the distribution of product variants of a complex mixture: a.. By altering cell culture process (upstream). Host cell proteins affecting specific modifications on recombi ant protein are first identified and modulators necessary to modulate those host proteins are then selected. Host proteins include enzymes involved in glyeosyiation, earboxyl alien, hydroxyiation, dearaidation, oxidation, C«ierminal sulfation, C~terminai carboxylase and amidation or any other posttransiational modification. Modifying the activity of these enzymes using small molecules, natural products, biologies, RNAi, R A, or DMA can be used for production of a recombinant protein with target modifications. A method that is capable of altering modifications on recombinant proteins are preferred tor use in the production of biosinular and hiohetter biologies than known systems thai knock-out. modificaions altogether. This method can produce recombinant proteins within target an es as opposed to knock out tec nologies which have oo possibility of targeting a desired modification range, b. During protein purification process (downstream) specific chromatography steps such affinity, ion exchange or mixed mode ehroraaiogaphy are used to remove specific product variants. Examples include but are not limited to removal of specific glyeosylation variants by lectin based chromatography, removal of certain charge variants such as deamidated and oxidized, specie by ion exchange and mixed-mode chromatography.

[0020] As with biosimdar development, the methodology described herein can be applied to other areas of biologic drug development. In particular, the disclosed methods have an application to situations where a production process for an originator biologic product needs to be changed. The key reason for a process change for originator recombinant proteins is to improve the cell line performance, to increase productivity and stability without changing modifications of said recombinant protein.

SUMMARY OF THE IN VENTION

[0021 ] The present invention provides methods for developing recombinant proteins with a fingerprint Hke similarity to reference products or originator products. The methods are particularly useful tor biosimilar development. The method includes five components (A.) analytical methods for measuring modifications on recombinant proteins (Bj in vitro and in vivo assays to measure biological activity (€) methods used or recombinant protein variant and structure activity relationship determination (D) cell culture methods for optimization of ceil culture conditions to produce the recombinant protein with the fingerprint level similarity to the originator and (E) purification methods to produce a recombinant proteins with the fingerprint level similarity to the originator.

[0022] Analytical methods for showing fingerprint similarity include

chromatography methods to separate and quaniitate different modifications as well as mass spectrometry methods to identify product modifications. The chromatography methods include but are not limited to size exclusion, ion exchange, reverse-phase, hydrophobic interactio chromatography, and released giyean analysis. Mass spectrometry methods including but are not limited to intact, mass and reduced mass analysis, peptide map and disulfide linkage analysis.

[0023] Biological activity is intrinsic to each recombinant protein being optimized. Frequently used bioassays used to test biological activity include but are not limited to; target binding ELISA assay, binding to cells expressing receptor, receptor internalization, receptor phosphorylation assays as well as assays that measure functional activity such as proliferation assays.

[0024] Manufacturing methods focus on optimization of cell culture conditions via addition of modulators) to growth media containing living cells that produce recombinant proteins. Addition of modulator^) to the living, cell culture medium can be used to reduce or augment the activity of specific .host protein(s) that control

modifications on the recombinant protein, which, may be a bi.osimi.1ar.. The modulators are selected to modulate the activity of host proteins .responsible for producing modifications. The modifications may include, but are not limited to, any of the following modifications; glycosylate, carboxylation, deamidation, oxidation, hydroxy!ation, O-sulfaiion, amidation, glycylation, glycation, alkyiation, aey!ation, acety!ation, phosphorylation, b.iotinylation, formyiation, lipidation, iodi. nation, prenylation, oxidation, palmitoylation, phosphatid iinos tol tion, phosphopanietheinyiatkrn, sialyiaiion, and selenoylatk , C- terminal Lysine removal.

[0025] Additional manufacturing methods can be used to obtain fingerprint like similarity on the recombinant protein being optimized. They include purification methodologies to remove undesired product species. .Examples include but. are not limited to removal of specific glycosyiation variants by lectin-based chromatography, removal of deamidated and oxidized charge variants such as deamidated by ion exchange and mixed- mo d e chromatography.

[0026] The present invention provides methods to identify, quantify, remo e, and assemble product variants to produce a biosimilar that exhibits fingerprint level of similarity to the originator.

[0027] In one aspect of the invention, there is provided a method for producing a biosimilar product showing a fingerprint level similarity to the originator; a. Establishing a relationship between product modifications and biological activity;

i. Identifying the number (n) of modifications present on a recombinant protein;

ii. Preparing recombinant protein variants enriched for one or two modifications at the time, at least at three different levels (high, medium, low) tor a total of 3B enriched variants produced;

in. Confirming the setoff modifications in the enriched

population using HPLC and MS based assays;

iv. Measuring biological activity of the enriched recombinant protein generated in ii). using biological assays relevant for said recombinant protein;

v. Establishing a relationship between the modification and the biological activity;

h. Measuring the quantity and type of specific modifications found on at least three ori inator batches using analytical assays;

c. Setting target profile ranges for the modifications of the originato based on data generated in b).

d> Growing living cells expressing the biosimllar with the identical amino acid sequence to the originator:

e. isolating the biosimi!ar from d) and comparing its modifications to the target set in h).

£ Selecting a plurality of growth media and one or more modulators to change modifications on thebiosimilar and growing the cells In the presence of said modulators. Modulators can be selected from the library of modulators; g. Isolating the product from f). and comparing its modifications to the target profile set hi c).;

h. Repeating steps f), g) with additional modulators and or at different

modulator concentrations to match modifications set in c). The modulators can be used alone or in a combination with each other. The set of exact modulation required to obtain the target profile provides a recipe for the production of said biosimilar and cell culture conditions are established to obtain th target profile. The target profile should not be set outside the specifications set for said originator;

i. Once the cell culture production process is optimized, isolating the optimized product, through a series of purifications steps which include but are not limited affinity, ion exchange or mixed .mode chromatography with a goal to remove specific product variants;

j. Measuring the quantity and type of specific modifications found on the biosimilar and comparing it to the target profile in c).;

L Determining product variants for each product batch using analytical data produced in h). and. in j},;

1. Comparing the type and quantity of the biosimilar product variants to the range of product variants produced by an originator;

m. Determining the impact of each product variant on biological activity based on the structure acti ity relationship and summing up the biological activity of all variants based on their relative abundance to identify whether the biological activity of the biosimilar is within the range for the biological activity the originator;

n. If specific product, vari.an.ts need to be removed, selecting a plurality of growth media and one or more modulators to change modifications on the biosimilar and growing the ceils in the presence of said modulators.

Modulators can be selected from the library of modulators. Isolating the product from n). through a series of purifications steps which include but are not limited to affinity, ion exchange or mixed mode chromatography with a goal to remove specific product variants;

o. Confirming that biological activity of the biosimilar is within 80 to 125% of the originator in in vitro and in vivo biological assays;

[00281 in another aspect of the invention, there is provided a method for a process change for an originator with a fingerprint level similarity to the reference Establishing a relationship between product modifications and biological activity.;

i. Identifying the number (n) of modifications present on a recombinant protein;

i . Preparing recombinant protein variants enriched for one or two modifications at the time, at least at three different levels (high, medium, low) for a total of 3n enriched variants produced;

iii. Confirming the identity of each enriched variant using

HPLC and MS based assays;

iv. Measuring biological activity for the recombinant protein variants generated in ii), using biological assays relevant for said recomb nant protein;

v. Establishing a relationship between the modification, and the biological activity;

Measuring the quantit and type of specific modifications found on the reference product or alternatively using product specifications to se the target profile range;

Growing living cells expressing the originator product in the presence of growth, media that produces higher liter or other beneficial cell line characteristics;

Selecting a plurality of one or more modulators to change modifications on the originator product produced using a new process and growing the cells in the presence of said modulators. Modulators can be selected from the library of modulators;

Isolating the product from d). and con-sparing its modifications to the target set in b}.;

Repeating steps d), e) with additional modulators and or at different modulator concentrations to match modifications set in. b}< The modulators can be used alone or in a combination with each other. The set of exact modulation required to obtain the target profile provides a recipe for the production of said comparable biologic. Target profile should not be set outside the specifications set for said originator; g. Once the ceil .culture production process is optimized, isolating the optimized product through a series of purifications steps which include but are not limited affinity, ion exchange or mixed mode

chromatography with a goal to remove specific product variants;

h. Measuring the quantity and type of specific modifications found on ihe originator product produced using a new prod ction process and comparing it to the target in h).;

i. Determining product variants for each product batch using analytical data produced, in b), for the reference product and in h). for the originator produced using a new production process.

j. Comparing the type and quantity of the originator product variants produced, using new optimized process to the range of product variants produced by the original process:

k. Determining the impact of each product variant on biological activity based on the structure activity relationship; adding the biological activity of all variants based on their relative concentration to identify whether the theoretical biological activity of the originator produced using a new process is within the range for the original process;

1. If specific product variants need to be removed, selecting a plurality of growth media and one or more modulators to change modifications on the originator produced using the new process and growing the cells in. the presence of said modulators. Modulators can be selected from the library of modulators; Isolating the product from n). isolating the optimized product through a series of purifications steps which include but. are not limited affinity, ion exchange or mixed mode

chromatography with a goal to remove specific product variants;

m. Confirming that biological activity of the originator produced using new process is within 80 to 125% of the originator produced using the original process; [0029] The method for optimisation may be used in conjunction with a bioreactor, shake flask or a wave bag or an other method known to one skilled in the art of process development. Assays selected for their ability to detect and measure- he presence of specific modifications are used to measure modifications. The assay module may be in liquid communication with the bioreactor for delivery of a recombinant protein to the assay module or can be carried out manually. The method can be implemented using a system having a library of individual modulators, which may be in liquid communication with the cell culture media and can be controlled by the assay module tor transfer of individual modulators into the bioreactor, a shake flask or other cell culture container.

[0030] The foregoing summary and detailed description -is better understood when read i conjunction with the accompanying drawings, which are included by way of example and not by way of limitation.

BRIEF DESCRIPTION OF THE FIGURES

[00311 Figure I contains the list of examples of host proteins and some of the known inhibitors.

[0032] Figure 2 is a schematic representation of a glycosylation pathway.

[0033] Figure 3 provides an example of a diromaiogram showing the carbohydrate peaks using the 2AB method of carboh drate analysis,

[0034] Figure 4 schematic of an antibody showing different antibody modifications and describing what are the product variants.

[0035] Figure 5 Schematic of the product variant determination approach

[0036] Figure 6 is a list of physi cochemical and in vitro biological

characterization assays for comparability assessment and fingerprinting. Example is for trastuxumah biosimilar.

DETAILED DESCRIPTION OF INVENTION

[0037] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not Intended to be limiting. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains,

[0038] in describing and claiming the present invention, the following terminology and grammatical variants will be used in accordance with the definitions set forth below.

[0039] The term ^• '"fingerprinting, " is a method of analysis of a recombinant protein that results in foil understanding of the product including but not limited to: a. All product modifications b. All produc variants e. Impact of product variants on biological activity (SAR equation)

[0040] The term "living cell," as used herein, refers to cell used for production of a hiosirm!ar version of a recombinant protein drug. Examples of a living cell include but are not limited to human, sheep, goat, cow, dog, cat, chicken, hamster, mouse, tobacco plant, and carrot sources. Examples of living cells which are commonly used to produce recombinant proteins as active drug ingredients include mammalian cells such as Chinese Hamster Ovary cells (CHO), murine myeloma NSC) cells, Baby Hamster Kidney (BHK) cells, SP2/0, 293, or CAP-T cells,

(0041 ] The term "host proteins ^" refers to proteins present in living cells, which interact with and modify recombinant proteins expressed in said living cells.

[0042] The term "modulators" include small molecules, biological compounds, natural products, lipids that can modulate the activities of host proteins that can be added to the solution containing living cells thai can specifically alter modifications on recombinant proteins. Modulators include both inhibitors and activators of host cell modification proteins. Modulator library refers ιο a collection of modulators thai can. be used to alter the activity of host proteins either to activate them or to inhibit them. The library of modulators may include small molecule drugs such as foeosyl transferase inhibitors, mannostdase inhibitor, biologic molecules such insulin, RNAt and UNA molecules, and other hiomoleeules known to those skilled in the art would recognize to affect post translations! modifications of recombinant proteins or their biosimilars being produced in host ceils,

[0043] In certain methods and em od men s one or .m re of the following compounds, known for purposes of this disclosure as Group I inhibitors, can be used to modulate modifications: 4,6,6'- h'ichloro-4,6,6'-irideoxy- i 2- isopropylidene-33',4 ^! -tri-0- aeeiylgaiaetos crose; hexa~0-bemoyl~4,6~0--isoprepylidenesucrose: methyl 4,6~diehlofo~ 4 _v 6"dideoxy ^» a-D-galaciopyranoside; methyl 2,3-di-0-tosy.l-4 _i 6-0-hen2ylidine-a-D- g ucoside; 6 ^>« chlorosucrose; 2 _> 44richkm>~2,3,44rideoxy-l\3',4 6 ¹ - fetraacetylgalaetosuerose; 4,643*be*¾yIidene~6 ^! -acetylsucrose; myo inositol bexaacetate; 3 _! 3',4 ^? _? 6 ^! 4etra4 -aa ylsucrose; 3^6,3\4\6'4¼xa43-aeetylsuero$e; 6,6 ^! ~diammo-6,6 ^! - dideoxy-sucrose D-giycero D-guloheptose; 2.3, 1 ^? ,3 ^! ,4' _l( 6 -hexa-0~acetyl~4,6~0- - dianhydro rehalose; 2,3 _. ,6 _} 3',4'-pema-0-acetyl-4 ch!oro-4-deoxy sucrose; l.,6~anhydro-3- nitro-3-deoxy-b-D-.gyk>se methyl 4,6-O-benzy!idene sophroside; sucrose 4,6, ,6 - tetratrityi 2.3 _> 3 ^> ,4 ^, 4etraacetate; 4,4 ^! ,6 ^, -trichioro«4,4',6 ^I rideoxygalactosucrose; 4 ,Γ,6'- te rachloro-4,6,1 ^ -tetradeoxysucrose; iricMorogalactosucrose 6 teriary butyl di phenyl sialyl; 2,3:4 _s 5-di~0-isopropyiidine~p-D-fi¾ctopyranose; trich!orogalactosuc-rose 3\4' lyxoepoxidc triacetate; 6' ch{oro-6'-deoxy-2,3,4 _J 6J' 3\4 -bexa-0-aceiylsucrose; 4,6,Γ,6'- tetra-0"trytyi-23,3 ^, ,4''-0-acetylsucrose 6 _; 6'"dichioro-6-6 ^I -dideoxysucrose: 3,4,6- triehloroglucose; isomaltulose octaacetate; 6~heriozyb _s 6 ^t ~ditosyi-2 _> 4 ^4 ^, -per!ta-0- aeeiyl sucrose; 2,3 dimethyl trichlorogaiactosucrose triacetate; 1 ^ iehloro- 1 ',6 -dideoxy- 2,3 _» 4 ₅ 6 _? 3 ^, ,4'-hexa-0-acety)sucrose; 6,6 ^?« di-0~tr tyl-2 ₅ 3,4 _? I ^* ,3\4 ^! ~hexaacetyi sucrose;

ociaaceiy! a D-eeOobiose; 6~chioro-6~d.eoxygalactose; 4, 1 ^f ,4',6'-tetxachloro-4, '^',δ'- tetradeoxy-2,3 _> 6,3 -tetra-0-acetylgalactosucrose; 6-O-acetyl- 1 ,2,-O-isopropylidine - -D- giueofuranose; 2,3 ₅ 4,6-tetra-0 rytyl glucose: 2,3:4,5-di-0~isopropyhdinefruciopyranosyl chloride; 4,6 Mric oro--4 _> 6,6-deoxy-3 ^, ,4 ^! -an.hydrosiierose; 6«chlo.n 6-deoxy- 2^4 r \4^6^e i ~0- ee ylsucros ; N-ociyl D-giueamirie; 2,3,4,64etra~G4rytyi glucose; r,2:4,()'-di4)-jse^ropyIidine--3,3 6 ^! ri-()"aeetyl sucrose; 2,3:4 _? 6-di-0- isopropyHdine-3 4\6'4ri-0-benzoyl~ -acetylsucrose; r,2:4,6-di-0 sopropylidke~3,4^di~ 0-acetyi~3 ^t ,6'-d.i-0-benzoy.lsucrose; r _> 2:4 _> 6-di-04sopropylidine~3 _> 3',4 ^, ,6 ^! -tetra-0- aeetytsuerose; 6-de«xy-6-catboxymetbyl- .1 ,2.3,4-tetra-O-trytyl glucospyranoside;

2 _! 3 _s 4,3 ^, ,4 ^, _s 6 ^, -hexa-0-acetylsncrose; 1 '^-dich!oro- 1 '^ -deo y-23 A6,3^4\6'-hexa-0- sucrose hexaaeetate; ,2,4,0~di~O-isopr0pyiidine sucrose; 3,4-aiiliydf o- 1 ,6-dichloro- 1 ,6 dideoxy-8"D yxo exofurattosyl^,0-anh^

3, 3 4 ' ^y -benzoyl sucrose; ieiraacetyl glucuronic acid; t ,2,3,4,S~penia }~acetykylitol; benzyl p-D-ihictop anoslde; 3 _s 3',4 ⁵ ,<V-teira-0-cyclohexarioyl sucrose; phenyl β-D- gaiaetoside; 2,3,4,6, 1 ,2,3 ,6-octa-O-acetylmaltose; 2,3,4,6 J '.S'^'- epa-O-acetyl sucrose; l \2:4 _ΐ 6-di-CMso ro !idi iε ^■ ~3 _f 6 ^, diacetyl sucrose; β-D ailose; 6'-chIoro-6'-deoxy sucrose; 6-0-methyi-4,1 6 richloro4 ₎ r,6 ^, -i-rideoxygalaciosucrose; 1 '^"di-O-tnesyW-O-benxoy!- 2,3 _! 6,3 ^' ,4 ⁱ -penta~0--aceiylsucrose; 6'-0-benzoyl-2 _s 3,6 _> 3',4 ^> -penta-0-ac€tyIsucrose;

2,3,4^, ^' ',3 ^, ,4',6'-hexa-0-mesylsucrose; Methyl 4,6 O-benzy!ifdene sophorose; Methyl 6- 0 rytyl~23,4"trl-0-benzoyl- -D-grucopyrai¾oside; & t~hutyldipheny1$il l sucrose;

1 ,2:3,5-di-0-phenyl-6-deoxy~6-thioacetyl~«~D~g!ucoforanose; 1 _i 3,4-tri~0-acet>4-6-chioro- 2,6-dideQxy x-D-ghicopyranoside: 6-C rytyl- 1 _> 2,3 _f 4 etra-0-acei.yl-a~D-glucopyranoside; 4,6~0-isopropyHdiBe-2 _? 3, r,3 ^, ,4' _! 6*4:vexa-0~berj2oy| sucrose; methyl 2,3-di-0~beuzoyl-4,6- di-O-mesylglueopyranoside; 4, .6 ^' - ricbloro-4J. 6'-rrideoxy-23,6,3,4-penia~0-ace yl sucrose; methyl 4 _> 6-0-benzyhdine-2,3-tU-0-tosyl- -D-allopyranoside; 2,3,4,6-tetra-O- rytyl glucpyranose; methyl 4 _> 6-0-beiizylidine-2,3-di-0-tosyl-a-D-gIucop 'ranoside;

Di-O-t ^yl^B^.S^'-he a O- cet l sucrose; 4,6; l ^, _s 2-di-0-isopro lίdine-3,3^4^ ^, - teira-G-aceiyl sucrose; l \2:4 _? 6-dK ⁾ sopropylidme sucrose; 6,3',4'~tri-()-aeetyi-4J ^, ,6 ^! ~ tr¾chlo5O~4, ,6 ⁱ -trideoxy galactosucrose; 6'~cbloro-6 ^! -deoxy sucrose; 7-O-tryt t 2,3,4,3,6- pem -0~acetyl-D-glycero-D-g«lo~heptose diethyl dithio acetal; 6'-chioro-2,3,4,6,l ' _f 3 4'~ hepta-O-acetyl sucrose; 3-acetarnido~t ,6-anhydro-2 ₅ 4-di-0-acetyl-3~deoxy β-D-gulose; Methyl 3~be?i2 Tiiido- ^> 4 _s 6-0-benzylidme ^» 3-deoxy-«-D~aliropyranose; 4, 1 ^ -trichloro- 4,r,6'~trideoxy galactosucrose (sucrafose); Methyl 3-aceiamklo-2,4~di-0--a.cetyl-3,6-- dideoxy-a-L-bexoside; methyl 2 _s 3-d -0-benzyl-4,6-di-0-mesylglucopyranoside D-ribo- 3 ₅ 4,5,6-ietra-0-acetyl-.l -niiro-hex-l -ene; 2-0-rrseihyI~D-glueose diethyl dithio acetal; Methyl 3-ace amido-2,4,6~tri-0~mesyl-- -D~mannosjde; D arabo-3,4,5,6 tetra-O-acetyM ~ mirobex- 1 -ene; 1,1 -diethyl sulphonyl-(2-0-tosyl-a-D-mbinopyranosyl) methane hydrate; Methyl glucoside fa urate; Methyl 2,3-anhydro-4,6-0~benzylidine-p-D- talopyrauoside; Methyl 2.,3-anhydro-4,6-0 ^« beu5f.ylidine-p~D-ta.k>pyranoside; 3-acetar do~ 2,4-di-O-acetyl- .6-anhydro~3 -deoxy-^-D ^opy.ranose; 1.1 ~d¾e(hylsu1phonyi-(3,4-0- isopropylidene-2~C osyl-ix-D-arabiaopyranosyl) methane hydrate; 2,3,4,5-tete¾-0- benzoyi galactose; D-mamK>-3 7-anhydix>-4-met oxy-5/ -isopropylidine~2,2~dieihyl sulphonyl, heptane; 2-acetamido- 1 ,2-dideoxy- 1 -rdtro-D -manitol; 1,1 -dieth [sulphony -L- ara ^' bo-2 ,4,54etraliydroxyhexane; 1 ' y-dichloro- 1 ; Methyl 3- ace†ams.do-3~deoxy-2,4.6~tri~0~aeetyl α-D-mannopyraiioside; Methyl S-besxscan do^^,- 0-ben¾ylidiBe~3-deoxy-2-0-mesyl ii ^; -D-aitropyranoside; Methyl 2-04osyl-4,6~G- benzy ideiie-a~D-gIueopyran side; 3 amino- 1 ^-anhydro-S-deoxy-p-D-altx'opyranose hydrochloride; Methyl 3-N-acetyl 3,6-dideoxy-2,4 di-O-acetyl- -L-roannoside; Methyl ",6"4etradeoxy raftlnose; 6,6 ^t 'dichloro-6,6 ^, ^idc«xy-3,4,3 ^, ,4 ^! -ieira-0-acetyI--sucrose; , 1 -diethylsulphanyl 1 -(a-D- yxopyranosy!)~n ethane; D-xylo-3,4,5,64etra-0- cetyl- 1 ~miro-hex-1 -ene; 1 ,1 - diethylsulphanyl - i -( ^' 2,3 A tj-i-0-aceiyl- -D-lyxopyranosy1>elhane; 2,3,4,6-tetra-Oaeetyj galaeiopyranose; 1 ~deoxy~ S. -niiro-D-glycerol-D-ga actoheptiiol; Methyl. 4,6-dlazido-2~( benzoyl-3-O-niesyl -D-giu«)pyt:anoside; 243 sopropy1.idi ii-3~acetai«ido-3>deoxy--a-D- ailofuxanose; 3,6~dideoxy~3-dimelhylamine--I..--n½.ni.iose hydrochloride; 3-acetimido-l ,2,4- iri"0-acetyl-3,6-dideoxy-|5-L-glucopyranose; 2 (NMPO(OPh)2)~3,4,6 triaceiyl gl eosazide; 23,6.3 ^, 4e raacelyl 4J '„4',6 ⁱ ietrach!oro 4, 1 ',4 ^! ,6 ^< tetradeoxy galaetosucfose; Axa inose diethyl mereaptal; 2~chioro-3-benzamino methyl hexaside; -04rytyi- 23 _! 4,6,3 ⁱ _! 4 ^, ,6'-hepta-0-aceiyisuerose: 2J '~0-diphenyl silane3,4,6 _s 3',4',<V-hexa-0-a etyi sucrose; 2 _> 3,4-trichloro-2,3 _s 4-trideoxy fructose; D-giycero-D-g !oheptose diethyl diihio aeetal; 1 L~2-0~methy1- 1 -ch ro-mositol pentahenzoate; Stevia glycoside; 4J ',6'- iriehiorotrideoxygaiaetosuerose tetraacetate OH-6 ^' ; sucrose ethyl 4,6-orth.oacetate hexaacetate; sucrose methyl 4,6~orthobutyraie hexaacetate; sucrose methyl 4,6 ^« orthoacetate hexaacetate; 4, ,6'-trihromo rideoxygalactosucrose pentaacetate; 6-0- beii2oyl-4 ₉ . ,6'-trichlorotndeoxygalaetosucrose tetraacetate; methyl 6-eh!oro-6~deoxy--a- D-galactopyranoside; methyl 4,6-dichloro-4-6-d deoxy- - ^' D-galac opyranoside; methyl 4,6-diehloi'o-4,6-dideoxy-a-D-gi eopyranoside; 3,&: ^' l.',4':3',6 ^> 4riajt-liydro-4-chioro-4~ deoxygalactosucrose; 3',6 ^, -anhydro-4,6, 4Tich1oro-4,6, 4ddeoxygalactosuerose; 4,1 ',6'- trich ^' lorogalactosuerose-3 ^, ,4 ^, 4yxoepoxide triacetate; 4,6 ^! - s.cbioro-4,6 ^, - dldeoxygalactosuerose hexaacetate; 4, ^? ,4',6'4etrach!orotetradeoxyga ^' {actosucn)i>e - dideoxysucrose pentaacetate OH-4; 4,6, 1 ',4',6 ^? -pentachloropeTrtadeoxygalaci.osucr0se inacetaie; 4,6,1 4 ^f ,6 ^! -peMachlor pentadeoxygaIactosorbos erose triacetate; 4,6,1 ',4',6 - pentac !oropentadeoxygaiactosucfose; 4,6,1.',4 ^, i ~

pentac!iloropetvtadeoxyga!ac osorbosucrose; 6-Q~aceiyM, i ^! ,6'- ribromc-4, 1 ',6- irideoxygalactosucrose; r _! 4';3^6 ^! -dianh dro-4~bromo-4-deoxygah t sucrose; 4~bromo-4- deoxy-D-galactose; 3 ,6-di~0-benzoyl- 1 _; 2~ 'isopropylidene- -D-glucofafaitoside 3,6-di-O- benzoyl- i ,2-0-isopropylidene~5-0-meti)yl- -D~gluco¾ranos; 6-chioro-6~deoxy-l ,2-0- isop.ropylidene-5-0-met.hyl- -D-glucofuranos; trans-l,2-0'ben2ylidene-D-glycerol; cis- 1 ,2-0-benzy!idene-D-g!ycei'ol; cis-1 ,3-0-beftzylideae-2-chloro-2-d.eoxy-D-g!ycerol; 4-0- mesyl-l V6 ~d.-0~tritylsjuerose pentaacetate; 6-chloro-6-deoxy-D-raai¾iono!actone; 6- ehloro*6-deoxy-D-manm>nolacione triacetate; methyl 2-aceiamido-2-deoxy-p-D- giueopyranoside; methyl 2-acetarnido-2-deoxy- ~D-g!ucopyranoside triacetate; me 2- acetamido-6--chl ro-2,6-dideoxy-^-D-g1ucopyranoside diaceiate; 4-0-Tnesylsuerose pentaacetate OH- 1 \6'; me 2-aeetamido-6-c1ilcHXi-2 ₅ 6-dideoxy- -D-glueopyranoside diacetate; -0-nicsyl sucrose heptaacetate; 3-0~acetyl-l,2:5,6-di-{)-isopropyiidene-a-D~ g!ucofuranose 3-0~acety!~ 1 ,2-0-isopropyHdene~a-D-glucoi¾ranose; 3~0~aeetyi-6~G ^» benzoyl -5-bromo- 1 ,2-0-isopropylide¾te-p-L dose; 3-0-acetyl-6-0-ber.i2oyt-5-ch!oro-l,2-0- isopropyik!eive-a-I>-gh.scose; 6~0-benzoyI~5-chior -l,2-0~isopropylidene-a-D~

giuc iuranose; meth l 2-acetamido-6--chIoro-2,6-dideoxy-a--D-glueopyranoside; 2-0- beBJZoyl-3-chioro-D-glyceraidehyde 2,4~dsniiropheny¾ydra2€me; methyl 4,6-0- benzylidene-2-cbloro-2~do)xy~«-D~raannopyranoside; methyl 3-0-bet zoyl-4,6-0- ben¾;y!idejte- ~D-glucopyranoside methyl 3-0-benzoyl~4,6-0-benzylidene~2 ^« chloro ^« ~D- mannopyrarioside; 2-chloro-2-deoxy-D-raaai¾tol; 4-(tetra-0-acetyl-P-D- glucopyranosyloxy)benza1debyde 6 -ch!oro-6'-deoxy~2 ₍ J':4,6-di-0-isopn.>py!idenes«crose; methyl 4,6~0-{p-mtrobeazyiidene)- -D-g!ucopyranoside diacetate; 4,6-0-{p- nitr benzylidene)- -D~glucopyra se triacetate; .methyl 4,6~Q~benzylidene~a-D~ ghicopyranoside diacetate; me 4,6-0-(Hi~nitrobenzylidene)-a-D-gl ^, ucopyrano.side diacetate (ax); &,6 ^! -dibromo~6,(>'-djdeoxysucrose hexaaeeiate; methyl 4,6-0-(ra-i«.trobe¾¾ylidene)--a- D-gl-ucopyranoside (eq); 6 _s 6'-diazido-6,6'~dkleoxysucrose; me 4,6~0-(m ^»

nitroben2y!idene)- -D-glucopyra«osjde diacetate (eq); 6'~hromo-6 ^< deoxysucrose heptaaeetaie; ^'- iamino-e^ - kieox sucrose; .methyl. 6-( -{n ni robenzyi)- ~D- glucopyranoside; 6 ^! -ai»ino-6'-deoxysuciOse; 6~eMoro ^» 6 feoxy-D-gkdiol pentaacetate; l,2~{ sopropyiide.ae~6-0-acetyl.> ~D-gl co&rfmose; 3,5-0~beBzy)idene~1.2~( - isop«3py!idene-6-0~acetyl- -D-gi co aranose; methyl 3-0~benzoyi-4 _> 6-0 ^« beiizylidene-2- cWoro- -D-glucopyran side; 6~0~trity1 -β-D-gi ucop ranose tetraacetate; 1 ,2,3,4-tetra-O- aceiyI.-(¾-D-giiicopyra.nose; 6-deoxy-6-tl«oro-p-D-g!uct>pyranose tetraacetate; 3,5- ben¾ylidene-l,2-0-isopropyHderie-a-D-glucofijranose; 6-de xy-6~ftuoro-D~gIue toI pentaacetate; methyl 2 _? 3,4,-tri-0-benzoyI--«-D--g!iicopyranoside; methyl 6-0-iosyi-a-D.- giueopyrarsoside; methyl 2 _s 3 _s 4-tri-0~acetyl~6-thio-6~S-acety]- -D~glucopyranoside; 6- chloro-6-deoxy-D-g!ucitoI (sy); 1 _? 2,3 _> 4-tetrc^0-acetyi-6-S"acety! ^» 6-thio-a~D-- glucopyranose; 1 ,2 ₅ 3 _i 4-teiTa~0-acetyl-6-ihio-a-D-glucopyranose dimer; 6~chioro~6~deoxy~ D-galactitoI; 6-chloro -6-deoxy-D-galactitol pentaacetate; 1 ^^.d-t tr -O-benzoyi-S^-O- isopropyhdene-D-matinitol; 3,4~0-isopr pylidene-D~Biannitoi; 1 ,2-0-isoprqpylidene-6-0- tosyl-a-D-giucoftiraiiose (crude); 2,5-di-0-benzoyl"i _> 6-dich!oro~3,4-0 sopropyIidene-D- mannitol; 2,5-di-O-benz yi- 1 ,6-dichioro-D-marautol ; .I ,2;3,5-di-0-benzyl.idene-6-0-tosy!- α-D-gj cofuranose; ^sS-S-di-O-benzy!idene-e-S-aeety!- -D-glucotliraiiose; methyl 2,3- arihydro-4 ₅ 6-benzylidene-ri-D-gu]op>ta«oside; 1.3:2.4: -5,6 ri-0-ediy!k1ei¾e-D-gluei†.o!; L3:2,4-di"0~ethylidenC"D-glucxto!; 5,6-anh.ydro- 1 _. ,3 :2,4-dk0-ethyiideiie- -giucitc-l;

l ₅ 2r5,6>di-( sopropy{idene-tt-D~gIucofurat3ose; .l ,2:5,6-di- -isopropylkiene-a-D- ail furanose; 1 ,2"0-is propyiidene~ -D-aIloiuranose; 6-chIoro-6-deoxy- 1 ,2-0- isopropyiidme~a-D-a!.lofuranose; 6-chk>ro-6-deoxy-D-all.ose; 2 ^4,0-di-0-isopropylidene sucrose tetraacetate; 1 ,2 : 5 ₅ 6-di-0-isopropyl idene- -D-guidfiiranose 1 ,2-0-jsopropylidene- ~D-g1ucof ran.ose; l,2-0-eyc hexyHdene~myQ~moshol; 1 ,2-0-cyc!dhexylidene-myo- inosi ol tetraacetate; 6~eMoFO~6<1eoxy~l 3,4,5,6- tetra~0-acety1-myo-inositol; 3,4,5,6-ietra-O-acetyl-myo-mositoi hydrate; 3 _s 4.5,6-teira~0- acetyl-1 "Ch!oro-1 -deoxy-scylio-inositol; myo-mositol hexaacetate; .1 -chloro-I-deoxy- scyHo-inositof pentaacetate; .1 ,2-dichloro- 1 ,2-dideoxy-myo-ittos tol tetraacetate; l-chloro ] -deoxy-scyl!o-inositol; 3*0-benzoyl- l ,2-5,6>0-di-isopropyiiderie~ -D~gluco&ranose; niethy! 6~chioro-6-deoxy«a-D-i¾ai)nopyranoside triacetate; 3 -0-benzoyI- 1 ,2-0- iSopr pyIidene-5,6~di~0~rnesyI~ -D~glacose; methyl 4,6~0-benzyIidcne- -D- raarniopyranoskle; methyl 2,3;4,{ -di-0-beji2ylidene- -D-mannopyraiia ^s 5ide; 6-chloro-6- deoxy~D~mannose; .methyl 4 _? 6-0-ber}¾y!iden.e~2-c. loro-2-dtn xy-tt~D-gIuc pyrano.s.ide; 3 _? 6~di~0 ^« be«zoyi- 1 ,2-0~isopropylidene-5-0-mesy{- -D-glucoiuranose; ό-0-benzoyl- 1 - chIoro iexar!~2,6~dioI (syrup):

fi^'-dichloro-e^'-dideoxy-D-tnaltose hexaacetate; 3O-acei>1-6-0- eftzoyl-i,2-0- i$opropyHdene~5~0~mesy!~tt~D-g!i}case; 3-0-aceiyi-5,6-di-0^e K yl~L2--0~isopropyIidene~ p-L d furaxsose; 5,6-di-O-benzoyl-l ^--O-isopropylidene-p-L-ido&ranose; phenyl 6-

i ,2"0-eihylene-p-D~fruciopyraaoside; 6 -chioro-6'-deoxysucrose; methyl 6-chloro-6- deoxy-«.-D- i«copyranQsid triacetate; methyl 2 _> 3-anhydro-4 _> 6-0-benzylidene- -D- allopyraxsoside; methyl 4 _> 6-0-beiizyiidene-2,3-di-0-tosyl-«.-D-g!ucopyraiioside methyl 4,6-0~benzylide.ne~ -D-al†ropyrai-!oside; L- 1 ^^iS^-peni -O- enzo l^-O-ro th l-chiro- inositol; 6-chl ro-6-dieoxy-a-D-alifopyranose tetraacetate; 3 _s 6-a«hydro- 1,2-0- isopropylidene-p-L-idofuranose 5-ehlor sa!phaie; 3,6-an.hydro- 1 _s 2 ^» 0- sopiopylid.ene-p~L- ido&ranose: 2-deoxyglueose; methyl 4 _> 6-0-benzylidene- x~D-ga ^' iactopyranoside; 4-chloro- 4-deoxy-D-galactitol; methyl 4,6-0-ben? Iidene-2 _> 3-di-0-tosyl- -D-galactopy.ranoside; methyl 4,6-0~benz-y!idene- -D-idopyranoside; 1 ,2-dichloro- 1 ,2-dideoxy-royo- inositol; Benzyl 2-aeetarnkio~4 }-(2-aeetamkk^

deoxy ^» 3,6-di-0-acetyl- ~D-g ^' lucopyraiiostde; 4'>chioro-4'deoxys crose hexaacetate OH-V 6-c¾loro-6Hleoxy-l,2-( isopropyhdefte~(i-D>ihictofurano8e; . ₅ 6'-dichloro-6,6'- dideoxysucrose pentaacetate QH-Γ; 2-chloroethyl β-Ό- iructopyranoside; 6 ^* hloFO--2,6" dideoxy- -D-g!ueopyranose triacetate; 4,6-0~benzylidenesucrose hexaacetate; 5,6- dichioro-S ,6-dideoxy- 1 ^-O-isopropylidene-j^-L-talofisrajiose; 5,6-dicMoro-5,6-dideoxy-P- L-talofuranose; Methyl neuraminic acid-S-acetyf-ehloride ethyl xanthate; Benzyl 2- acetamido-3 _i 6-di-(}-benzyl~2-deoxy-4-0~(3,4 ₅ 6-tri-0-beu^y!~B'D-ma

giucopyranoside; Benzyl 4-0-p-D-galactopyranosyl-p-D"glucopyranosk1e heptaacetate; Benzyl 2-ac€tam do-4-0-(2-aeetaroido-2-deoxy~P-D~gl c<^yran syl)-2-deoxy-p-D- giucopyranoside: Benzyl 2-acetamido-3 >-di-0-benwl-2-deoxy-4-0-(3,4 _> 6 ri-0-benzyl-3- D--arabinohexopyran-2"idogyl)-tt~D-gl copyranoside; Ethyl-4 _s 6-0-benzyHdene-2~deoxy-2- phthlamkio- 1 -thi -p-D-glueopyranoside; 4,6:2,1 ^! ~di~0~isoptopylidenesucrose tetraacetate; S^'^'^'-tetra-O-acet lsucrose; 3^4 ^! -di-0~acetyh4J ^6' ri.chlorotrideoxygalaciosucrose; methyl 4-eWoro-4-deoxy-a-D-galactopyranoside; 3 J \4\64etrachIoro--3, 1 ^! ,4\6'~ ietradeoxyallosorhosuerose; methyl 6-chloro-6-d oxy-<x~D-gkcop an side;

ga!actosucrose; 1 \6'-dichloro- 1 '^'-dideoxysuerose hexaacetate; 6,6 ^s -diehloro-6 ₅ 6'- dideoxys erose tetraacetate OH-2,1 2,3- -isopropyhdene-6, 1 ' ₅ 0 x Mniylsucrose triacetate; 3"0~ac«tyl~3\6 ^! ~di ^« 0-benzoyi-4,6:2 _> -di-0-isopropylidenesucrose; 4,6:2, Γ-di-O- is pfopyiideuesucrose tetrahenzoate; 4, 1 4-0- mesylsucrose heptaaeetate; 3-acetaiBido-5,6-di-0-acetyl~l _f 2 sopropylidene- «D~ aMof ranase; methyl 2~acetamido-3 ? ⁾ -acetyl-4,6-d -0-mesyl-tt-D-gluwp> ⁱ T¾isoside; methyl 4 ₄ 6~0-benzylidene-2 _< 3-immO'-a-D-maanopyranoside; methyl 4,6 3-beazylidene~ 2,3~imino4N -p-nitrobenzoyl ~«4 aik>$ide; Taethyl 3-aceiaiBid -4,6-0-benzylidene-2-0- mesyi-a-D-aitropyranosid; methyl 23-aiiiiydro~4,6-0-benzylidette^-D-ialopyranoside; methyl N-acety1-4 _r 6-0-ben2ylidene-2 ^inb\o~a-D--mannopyanoside; methyl 4,643- benzvHdeae- -D-sophoroside tetraacetate OH -3; methyl 2-0"beazoyl-4,6--0--benzyiidene- a-D~gliicopyranoaide; Ethyl-3-0-ben^y1"2-d xy-2~pbthlam.ido- 1 ¾ίο-β-0~

glucopyranoside; methyl e^ -dichloro^^ -dideoxy-P-D-cellobioside: methyl 2 -di-O- aeetylr4-0-mesyl-6-ihiocyanaio- ~D-ga{actoside; methyl 3-aceiarnido-3-deoxy-2,4,0-tri~ O-mesyl- -D-glueopyranoside; Me N-ac€ty!~4 6-0-bi¾izyKdeae-2,3-dideoxy-2,3-imini tt- D-alloside; Me 4,6-O-ben2y1idet\e-2 -ia ^, d«0~N-{2 _J 4-diT«tr pheayl)- -D-alloside; lactose octaaceiate (a/B); Chitobiose oxazoline hexaacetate; hexadecvl 3^4'4}-isopropylidene-p- D-laetoskle; methyl 4,6-0-isopropyIideae-{ -D-g1ucopy.ranoside; hexadecvl β-D-lactoside; tetracosyl β-D-lactoside; methyl 3~deoxy~3-fiuoro-4,6-0-isopropylideae-p-D- allopyranoside; methyl 3~deo -3-i1uoro-p-D-:aliopyranoside; 2-deoxy-2~.fluoro- 1 ,3,5-tri~ 0-(4-ehlorobeazoyl)~a-D-ribofuraaose; p-Mephenyl 2-azido~346-tri-0-p~chlorobeazyl- 1 - thio-^-D-galactosid; hexadecvl β-D-Iactoside pentaacetaie OH-3\4'; methyl 2,3,6-tri-O- henzoyl- -D-galactopyran side; AHyl-p-D-ch tobioside; trichl r ediyl 2~aeetamido-2- deoxy- -D-glucopyraiioside triacetate; frich!oroethy! 2-aceta ido-2-deox.y- 4

glucopyranoside triacetate; tee 2-acetamido-3-benzoyi-4,6-orthoacetyl^-D- g eopyranoside; iriehlofoeihyl β-D^chitohioside heptaaeetate; {2\2' _s 2' rjchloroethyi) 2- acetamido~2-deoxy"3~0-ben2oyl-6~0-acetyl~p-D~glucopyranoside ; aliyl β-D-chitobioside heptaaeetate; 3 _s 4,6-tri-0-ben2yi-D-maaaos.e; tetr -O-hen oyl a-D-glueopyranasyl bromide; tetra-O-beazoyi-2-hydroxy-D-giucal; 3,4 i4ri-0-ben¾oyl- -D-bexopyranos-2- losyl bromide; benzyl a- D-mamx>{ 1 3 )bioside 6-ehioroaeetate hexabenstoate; benzyl - D-manno( 1 «3)bioside 6-OH hexabenzoate; 2~deoxy-2-p haiimidO"|¾~D-glucosamine tetraacetate; 4~deoxy-4-fluerc>~D-galaciose benzyl 2-acetas«ido-2-de xy-a-D- glucopyranoside; benzyl 2-acetaroido-4 _; 6~0-benzyiidene-2--dei!xy~a-D--g!uci3side; benzyl -D-mamiop Tanos.kle; EthyI-6~(i~acetyl~3~0-benzyl~2^ieoxy-2^^

glneopytanoside; benzyl 2~acetamido-6-0-acetyl-3--0-beiizoyl--2-de0xy- -D-glucoside; benzyl 2-aeefamId ~3-0-benzyl-4 _i 6 )-benzyIidei)e- -D-g)i¾coside: EiS 2-0-{2- acciamido-p'D-g ucopyranosylJ- -D-mannosid hexaacetate; Benzyl 2,4-di-benzoyl-a-D- mangopentaoside tetradecaacetate; Benzyl 2,4-di-0-benzoyl-3-0-[2-{H2-aceiamidiv2~ deexy-SAb-in-O-acetyl-p-D-g!ueopy^^

D-ffianiiopyranosi.de: Benzyl 2-aeeianud<>-3-0-{tetra-0-aceiyH3- -ga1actopyranosyiH,6- 0-benzyIidene-2-de»xy-a-D-giueopyranoside; Benzyl 2-acetan¾ido-3-0-(tetra-0-8cetyl-^- D-galactopwanosyl)-2-deoxy-«-D-gl-ucoside; L ^' 2:5,6-di-0-isopropylidene- -D- galactofuranose; 2-0~acetyl~3,4,6-tri-0-benzy1-D-gliicopyranose; Benzyl 2-ace†afiiide-4~ O-^-O-ac tyWAe-tri-O _^ ben l-p-D-gluc^^

giucopyranoside; benzyl 2-acetamido-3,6-di~0->benzyl-2>deoxy-u-D-gkicopyranost de

deoxy- -D-glucopyranoside; 2-0-P~D-g!iicopwaiiosyl~D--glueop Tanase; Benzyl 4-0(3,4- 0-isopropylidene-p-O-galactop> anosyl)-p-D-glycopwanoside; 2-0-a-D-mannopyianosyl- 3 A^-iri-O-benzyi-D-mannopyranose; 4-methyiphenyl 1 ~fhio-p Mactoside heptaacetate; 4-roeihylphenyl 4-0-{2,6~di-0-acetyl-p-D-galae^^^

giucopyranoside; 4- ethylph.enyl 4 ^« 0~(3 _> 4~0 ^« isopropylidene- -D~galactopyranosyl)- i - thio-p-D-glncopyranoside; Ethyl 3~0~benzy!*2-deoxy-2-phthalimido-4-0-P-D- galactopyranosyi-l-thio- -D-g!ucoside; Ethyl 2~aeetamido~6~0-8-cety] ^» 3-0-allyl-2-deoxy~ 4-0-(ietra- -aeety!-p-D--galactopyranosyl) 1 -ihio-p-D-glueopyranoside Benzyl 2- aeetamido-6-(^ac«tyl~3-0~benzyl~2-deoxy-a-D-glucopyran.osid e; Benzyl.2 _* 4-di-0-benzoyl- 6-0-(tetra-0-benzoy t-D-ma3inop>^anosyi}- -D-iTiannopyraiioside; Benzyl 2 &c*3amido* 6-Q-aceiyl-2~deQxy~3-0-(ietra~0~acetyl~p-D~gala^^

Benzyl 2-aci^niido~6~O-acet -3-0 tet^

benzyi-a _--f cop>Tai)osyI)-2-deoxy~ -D-glucopyranaside; l,4,6-tri-0-ace yl. ^» 3-0-(tetfa-0 ^« acetyl- -D~gaIactopyranosyl)~ -D~gaiaciopyranose: I ₅ 4 _s 6-tri-0-acetyl-2-O-(tri-O-bettzyl- - L~iueopytanos> )-3~0-(teta^O~^ Benzyl 4 _; 6-0-benKvHdene- ~D-glucopyranoside; Benzyl 2,3-di»0-beazyl>4,6-0-benzyHder5e-a-D- giucopytanoside; Benzyl 2,3-diO-benzyl-a~D-ghic pyranoslde; Benzyl O-o-D- galactopwanosyHl ~~*4)-2 _P 3-di-0-ben2:yl-a-D- glucopyranosule; Benzyl 2-aeei.am.ido-3 ^« 0-be«zyl-2 _> 6~dideoxy-6 ^« iod -a«D- glucopyranoside; Benzyl 2--acetaffiido-3-0-benz>^2,6~dideoxy~o-D~glucopyranosi Benzyl 2-acetamido-6-0-aceiy1-3-0-benzyl-2Hk y <-D-g!ucopyranoside; Phenyl 2,3,4,6- ietra-O-a etyl- 1 -thio-a-D- annopyranoside; I J^d-ietra-O-acetyl-p-D-raanniipyranose; ½3,6-fetfa-0 enzeyl^^

gal.actopyranosy1)-a & β-D-glucopyranose; l ₅ 23,6- etra-0-benzoyl-4~0~(2 -di-0~benzoyl- p-D-galaetopyranosy -p-D-grueopyranose; l _> 2,3,6~tetra-O~benz0yi-(2,3,6-tri-O-benzoyl-|¾- D-gala.ctop Tanosyl)*P-D~glucopyranose; l ,2J _? 6-tetra-0-benzoyl-4-0~(2.3--di-0~ben2oyl-f¾-- O-galaetopyi-anosyl)-a-D-glucopyra.nose; l,2 _> 3 _J 6-tetra.-0-benzoyi-(2 _s 3 _> 6-tt 0-beji2oy!>P- D-galactopyranosyl;)-a-D ^« glucopyran.ose; Phenyl 2,3,0~iri-04>enzoy1-i 4hio-p~D- gajaeiopyrarjoslde; Phenyl 3,6-di-O-benzoyl- 1 hio ^« j¾-D-galactopyra«ostde; Phenyl 1 -thio- β-D-gaIaciopyranoside; Benzyl 4-0-{4-6-0-4-methoxybenzyIideiie-p-D~ga!aetop>Tanosyl)- β-D-glucopyranoside Benzyl 4 )-{2,3-di-O-ace(yl ^« 4,6-O->4->methoxybenzyKdene- -D-- galaetopyran.o$yl)~2 _? 6 ri"0-aceiy!-p--D-glucopyranoside; Benzyl 44 ^" H ^' 243~acety]-3,4-0- isopropyiidene-6 } ^« 4~metl^

giiicopyranoside; Benzyl 4-0-{2-0 ^» ac€iyi-p-D-gafaciopyTanosyl}-2 _i 3,6-tri~0-acei.yl«P-D- gl copyranoside 2,3,6,3 ^, ,4'-penta-0-aceiyIsucrose; (4~methyl phenyi)sulphenyl 2-azido- 3A6- ri-0-i4-c¾lorobe^yl)~2-deoxy-j¾-D-galactopyrajioside; 4,6-0-<4- methoxybenzylidene)-2-acetamido-2-deoxygalaciopyranose; Benzyl 2-aeetarnido~2- deoxy-3 _i 6-ds-C}4?enzylHi-D-g!ueo yranoside; Benzyl

ga!aefopyranosyl)"p"D-glucopyranoside; Benzyl 2,3 _i 6-tri-0-benzy!-4-0-(2 3-di->0>benzyl- 4,6-0~benzy]idene~p-D-galaclopyranosyl}--p--D--glucopyranosi de; Benzyl 23 i-t -Q- benzyM-0-(2 ,64ri~O- eiizyl ^ Ethyl 4,6-0- ben¾y.lidene-2-deoxy-2-phthaIirnido- 14hio4 galactopyranoside: Benzyl 2,3-di-0- benzyl-4,6-0-benzyIldene-p"D-gaiaetopyranoside; Benzyl 2,3-d -0»ben2yl~4 _> 6»0~ benzybdene-p-D-galaetopyranoside; 3-0-(2-acetamido-2 ^« deoxy-tt-D-galactopyranosyl)-D- galactose; 3-0-(2 ^> aeetaffiido-2-deoxy- -D-galactopyfattOsyl)-D-galact<.>se l ,3,4 ₅ 6 etra-0- aceiy!~2~d£oxy-2-phi a[n«5di D-g! ucopyranose; Methyl 3 ,4,6-tr.i-O-acetyl -2-deoxy-2- pht allmido-p-D-ga!actopyran side; Methyl 4 _s 6-0-benzy1k ^' lene-2-deoxy-2-phihaH ^' mido-3- 0-(3 ^-tri-O-acetate-a-galactopyraooside- 1 ,2-orthoacetyl )-$-D-gaIaet©pyranoside : Methyl 4,6-0 ^« benzyHdene-2-deoxy~2-phthalimido-j¾-D~galactopyranosi de; 1 ,2 ,4,6~tetra~0> acetyl^ 2,3, 4,6 et a-0- ce^^ Thiophenyl 2 _¾ 4,6-tetra ⁾ ~be»2yhp-D-'galactopyraaoside ^* 2,3 _> 4,6~tetra~0-ben2y!-D~galactose Methyl 2-chioro-3-aeetaraido-2 _> 3~dideoxy-a~D-altfopyraaoside Methyl 3-aceiaitMdo ^» 2,3 ^» dideoxy- 4,6-isoprpyl3cleiie-a-D"giuc0p Tanoside; Methyl 2,3~anhydrodideoxy-2.,3~acetamido-4 _> 6- 0-benzylIdene-a-D ^» aliopyraitoside; Methyl 2,3~dideoxy-3-acetaiiido-4,6-di-0-mesyi-a~ D-glucepyn oside; Methyl 3-sn inohydroeh!oride-3-deo y~4,6-benzylldene~a-D~- manooside; 2 '-iseprpylidene~2\3\4 ^, ii-0-aeetyI sucrose; Methyl a-D-ga!actoside;

G amrna-D-G al aetonoi aetone .

[0044] The term "recipe" refers to a mixture of the modulators and their concentrations that will be used to produce said recombinant protein or b osimilar with the target, profile.

[0045] The term "recombinant protein" refers to any protein species, produced in living cells., systems, or organisms resulting from recombinant DMA technology. As •used herein, the term "recombinant protein" includes but it is not limited to, proteins, polypeptides, and monoclonal or polyclonal antibodies and their biosimilar versions.

[0046] As used herein the term "antibody" encompasses whole antibodies including single chain antibodies, and antigen whole antibodies, and antigen binding fragments thereof. Fab, Fab' and F(ab')2, Fd, single chain Fvs (scFv), single chain antibodies. disu!fide-Iinked Fvs (sdFv} and .fragments comprising either VL arid VH are all within the present definition of the term "antibody. " Antibodies may originate from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken.

[0047] The term "biosimilar" refers to a recombinant protein, commonly with identical amino acid sequence to a reference commercial product that contains, similar, very similar to or same posMranslationai modifications as the reference product yielding similar biological activity to that product. [0048] The term "reference product" refers to a currently or previously marketed recombinant protein, also described as the "originator" or "branded product" serving as a comparator in the studies. An "originator" or "branded" product are- examples of a reference product.

[0049] The term "reference standard" refers to a highly characterized drug substance. The reference standard s prepared during the drag development cycle to serve as a comparator to all subsequent lots being manufactured.

[0050] The term "hiobe ter" refers to a version to an original biological drug with the same protein sequence but post-translational modifications that are outside the target profile range, which affect the drug's biodistnbution, pharmacokinetics and pharnu¾cod>¾ainks.

[00511 As used herein, the term "candidate" with reference to biosimilar drug or bio-better drug, refers to the intent that if will be the subject of an application for commercial sale submitted for approval by one or more drug regulatory agencies in one or more different jurisdictions.

[0052] Recombinant proteins generally contain post-translational modifications.

These modifications include but are not limited to: glycosyiation, carboxylation, hydroxylation, Q~suliaiion, anndaticn, glycosyiation, glycation, alkylation, acylation, acetyiation, phosphorylation, biotinyiation, formylation, iipidation, iodinaiion, prenyiation, oxidation, pahtutoylation, pegylatlon, phosphaiidylinositolation,

phosphopantetheinylation, siaiylaiion _; and selenoylation.

[0053] The term "glycosyiation" refers to attachment of oligosaccharides to proteins and represents the most commonly found post-translational modification of recombinant, proteins. Oligosaccharides consist of monosaccharide units that are connected t each other via gfycosidic bonds. Oligosaccharides may also be branched, with each of the sugar units in the saccharide serving as an optional branching point. The oligosaccharide chains are attached to proteins co-translationally or post-translationaHy, via specific asparaginic ( -linked) or serine/threonine (0-linked) residues, For N-linked. glycosyiation the consensus amino acid sequence of recombinant protein is Asn-X- Ser/Thr. 0-sulfation entails the attachment of a sulphate group to tyrosine, serine and. threonine residues mediated by suliblrans erases. Armdation is characterized by the replacement of the C- em inal carboxyl group of a protein with an amide group, y~ carboxylafion and -hydroxyiaiion modifications are mediated by specific carboxylase and hydroxylase enzymes, with conversion of target glutarnate residues toy- earhoxyglutaniate (Glu—— Ok) and either target conversion of aspartate residues to - bydroxyaspariate (Asp—— + Hya) or asparagine residues to -hydroxyasparagine (As« ÷ H n).

[0054] The phrase " modifications on the recombinant protein are substantially the same as the post-modifications on the .reference protein" can he taken to mean that the levels of post-tran iaiiona! modifications are within the ranges of the post- translation modifications identified in at least five lots of the reference protein.

[0055] The method for developing "target profile" and 'target profile range" or

"target range ^" is described in Examples 1 and 2,

[0056] The disclosed method involves developing a media recipe from growing cells to produce a recombinant protein of interest. The media can be any medium that .is appropriate for growth of the cells that are used to produce the recombinant protein.

[0057] The media can include supplements of which concentrations may be known or unknown. Examples of suitable supplements include salts, amino acids, vitamins,, lipids, gluta ine, glucose and galactose. Growth media lor cells can be made custom or purchased commercially from companies like Gibeo, Lonza, Millipore, H ^' yelone, GE and others familiar to those skilled the art of upstream process media development,

[0058] Any ceil that can be used for the production of the target recombinant protein can be used in the present method. Suitable cells generally will excrete the produced protein into the medium from which the recombinant protein can be isolated. Most commonly used cells are all variants of CHO cells, CAP-T cells, murine myeloma NSO cells, Baby Banister Kidney (BRK) cells, SP2/0 ceils, 293 ceils or NSO cells.

[0059] The cells can be grown as a batch, as in shake flasks, or in any type and size of bioreacior and/or wave bags for production of the recombinant, protein.

Manufacturers of growth chambers and apparatuses include but are not limited to those produced by Mi Hi pore. General Electric, Eppendorf (New Brunswick), and Sartorius Steadim.

[0060] When cultured in a bioreactor, a control mechanism for altering conditions for production of the recombinant protein may be also provided. The mechanism for altering conditions may be in digital data communication with the controller so that an operator may alter production conditions by providing input to the controller. Conditions which may be altered using the controller include, but are not limited to: temperature, pressure, gas flow, agitation, and composition of growth ^• medium components. Examples of growth medium components include, but are not limited to carbohydrates, salts, proteins and lipids and one or more components from the modulator library.

[0061 ] Any modification that can be controlled by the addition or removal of a modulator is amenable to modulation by the present methods, Glycos ^y lation is an example of a modification that is particularly amendable to the optimization by the present methods as the host proteins involved in the glycosylation pathway are well, known {Figure 2) and can be modulated by a variety of inhibitors (Figure 2). Other modifications are described in the definition section.

[0062] Any suitable method known to one skilled in the analytical arts can be used for measuring the levels of modifications. Mass spectrometry (MS) is a powerful method for analysing and quantifying modifications. Some of the MS based methods amenable to said analysis may include but are not limited to: intact mass analysis, reduced mass analysis, peptide map analysis, and disulfide linkage analysis. Intact mass analysis by ESI-MS is used for identification and quantitation of modifications on a recombinant protein including but. not limited to. glycosylation and€ -terminal lysine content. To analyze complex molecules such as antibodies, reduced mass analysis and peptide mass analysis should provide detailed information including the exact amino acid that has. been modified. To conduct reduced mass analysis heavy and light chains of the antibody are first reduced, then resolved using reverse phase chromatography or other methods known to one skilled in the art and subsequentl analyzed using ESI- S, To conduct, a peptide map analysis, an antibody is fir digested with an enzyme that leads to antibody fragmentation. Each peptide is first resolved on appropriate

chromatographic media and then analyzed by ESI- MS for amino acid sequence and modification such as glycosylatkm, dea nidaiion, oxidation, disulfide scrambling, and C- terminallysine content. Enzymes thai cars, be used for recombinant protein digestion include but are .not. lim ted to ^' trypsin and Lys-C.

[0063] Chromatography by HPLC or UPLC is another powerful method to analyze recombinant proteins. For example, glycan species can be quaniitated using a fluorescent 2AB labeling method. In this method, glyeans are first removed from the protein by digestion with -glycanase and then a fluorescent label is added to each glycan. The glycans can then be resolved using HILIC based chromatography and quaniitated by measuring relative area under the curve. For oxidation quantitation an HIC based method can be used.

[0064] To determine the level of dearnidaiion using chromatography based methods 1SOQUANT Isoaspartate Detection Kit can be used. The ISOQUANT

Isoaspartate Detection Kit uses the enzyme Protein Isoaspartyl Methyl transferase (PIMT) to specifically detect the presence of isoaspartic acid residues on a recombinant protein. PIMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to isoaspartic acid at the a-carboxyl position, generating 8-adenosy3

homocysteine (SAH) in the process, SAH formation is then quaniitated in the sample by comparing it to the standard provided in the kit.

[0065] The present invention provides methods to identify, characterize, quantify, remove, and assemble product variants to produce a biosimiiar thai exhibits fingerprint level of similarity to the originator.

[0066] In one aspect of the invention, there is provided a method .for producing a biosimiiar product showing a fingerprint level similarity to the originator as follows:

(a) Establishing a relationship between product modifications and

biological activity;

i. identifying the number (n) of modifications present on a ■recombinant, protein; it. Preparing a recombinant protein enriched for one or two modifications at the time at least at three different levels (high, medium, low) for a total of 3n enriched variants produced;

iii . Confirming the identity of each enriched variant using

HPLC and MS based assays;

iv. Measuring biological activity of the enriched recombinant protein generated in ii). using biological assays relevant for said recombinant protein;

v. Establishing a relationship between the modification and the biological activity;

(b) Measiiring the quantity and type of specific modifications found on the at least three originator batches using analytical assays;

(c) Setting target profile for the modifications of the originator based on data generated in b),

(d) Growing living cells expressing the hiosimiiar with the identical

arn noacid sequence to the originator;

(e) Isolating the biosimilar irom d) and comparing its modifications to the target profile set in c),

(i) Selecting a plurality of growth media and one or more modulators to change modifications on the biosimilar and growing the cells in the presence of said modulators. Modulators can be selected from the library of modulators;

(g) Isolating the product from f). and comparing its modifications to the target profile in e},;

(h.) Repeating steps i), g) with additional modulators and or at different modulator concentrations to match modifications set in b). The modulators can be used alone or in a combination with each other. The set of exact modulation required to obtain the target profile provides a recipe for the production of said biosimilar. Target profile should not set be outside the specifications set. for said originator; (i) Once the cell culture production process is optimized, isolating the optimized product, through a series of purifications steps which include but are not limited affinity, ion exchange or mixed mode

chromatography with a goal to remove specific product variants;

(I) Measuring the quantity and type of specific modifications found on the biosimilar and. comparing it. to the target in h).;

k) .Determining product variants for each product hatch using analytical data produced in b), and j).;

(!) Comparing the type and quantity of the biosimilar product variants to the range of prod ct variants produced by a originator;

(m) Determining the impact of each product variant on biological activity based on the structure activity relationship; summing up the biological activity of ail variants based on their relative concentration to identify whether the biological activity of the biosimilar is within the range for the predicted biological activity the originator;

(n) !f specific product variants need to be removed, selecting a plurality of growth media and one or more modulators to change modifications on the biosimilar and growing the cells in the presence of said modulators. Modulators can be selected from the library of modulators; Isolating the product from n). through a series of purifications steps which include but are not limited affinity, ion exchange or mixed mode chromatography with a goal to remove specific product variants;

(o Confirming that biological activity of the biosimilar is within 80 to 125% of the originator in in vitro and in vivo biological assays;

[0067] hi another aspect of the invention, there is provided a method for a process change lor an originator with a fingerprint level similaiity to the reference standard;

(a) Establishing a relationship between product modifications and

biological activity;

L Identifying the number i n) of modifications present on a recombinant protein; ii. Preparing a recombinant protein enriched tor one or two modifications at the time si least at three different levels (high, medi m low) for a total of 3n enriched variants produced

iii. Confirming the identity of each enriched variant using

HPLC and MS based assays;

Vs Measuring biological activity for the recombinant protein variants generated in ii). using biological assays relevant for said recombinant protein;

V, Establishing a relationship between the modification and the biological activity;

(b) Measuring the quantity and type of specific modifications found on the reference product or alternatively using product specifications to set the target range;

(e) Growing living cells expressing the originator in a presence of growth media that produces higher titer or other beneficial cell line

characteristics:

td) Selecting a plurality of one or more modulators to change

modifications on the originator produced using a new process and growing the cells in the presence of said modulators. Modulators can be selected from the li brary of modulators;

(e) Isolating the product from d), and comparing its modifications to the target set in b}.;

(f) Repeating steps d), e) with, additional modulators and or at different modulator concentrations to match modifications set in b). The modulators can be used alone or in a combination with each other. The set of exact modulators and concentrations required to obtain the target profile provides a recipe for the production of said comparable biologic. The target profile should not be set outside the specifications set for said originator; (g) Once the cell culture production process is optimized, isolating the optimized product through a series of purifications steps which include but are not limited affinity, ion exchange or mixed mode

chromatography with a goal to remove specific product variants;

(h) Measuring the quantity and type of specific modifications found on the originator produced using a new production process and comparing it to the target in b},;

(i) Determining product variants for each product hatch using analytical data produced in h). for the reference product and in h). for the originator produced using a new production process,

(i) Comparing the type and quantity of the originator product variants produced using new optimized process to the range of product variants produced by the original process;

(k) Determining the impact of each product variant on biological activity based on the structure activity relationship; adding the biological activity of all variants based on their relative concentration to identi y whether the theoretical biological activity of the originator produced using a new process is within the range for the original process:

(1) If specific product variants need to be removed, selecting a plurality of growth media and one or more modulators to change modifications on the originator produced using the new process and growing the cells in the presence of said modulators. Modulators can be selected from the library of modulators; Isolating the product from. n)„ isolating the optimized product through a series of purifications steps which include but are not limited affinity, ion. exchange or mixed mode

chromatography with a goal to remove specific product variants;

(m)Conil rming that biological activity of the originator produced using new process is within 80 to 125% of the originator produced using the original process;

[0068] The described method results in the development of a .recipe for media ha ving concentrations of a variety of modulators that are required to produce recombinant proteins matching a target profile. The recipe is ideally used to produce the recombinant protein after a manufacturing process change or during biosimilar development. The .method is particularly useful in the development of biosimilar products having

modifications that are difficult to match and have the advantage that they can be used while keeping cell productivity high because the method decouples the productivity from target profile. Examples where the method can be used include trastuzumab biosimilar.

EXA PLE ΐ SETTING A TARGET PROFILE

[0069] Tins example demonstrates one method, for identifying a target profile for development of a recipe for production of a recombinant protein. In order to identify target, profile or target profile range, at least 3-5 batches of the original reference product should be examined for the type and the amount of specific modifications. For biosimilar development a reference is defined as reference product. For a process change, a reference is defined as one batch of the reference standard and an additional 4 batches of the produc made using the original process. In the example below to set target modifications for biosimilar development, 5 batches of the reference product were analyzed for

modifications. Out of 14 modifications, two modifications (giycosylation-- GO and giycosylation G2 were not observed. Other modifications were measured and are shown in Table 1 to he present at different levels on different batches. To set the target profile, first the exact measurements for each modification are identified for all five batches 1-5. For example, for Giycosylation -GO glycan, the 2A.B g!yean analysis showed variability from 2-6%. To set the target profile, the range is extended by 1% on the lower limit and 2% on the upper limit yielding a target profile range of i %-8%. Using this method target is set. for each modification.

Table 1 Setfiag Tswrgei Profile

FT M Batch \ Batch 2 Batch 3 Basel; 4 Basc 5 Targei

Profile Range

Glycosylates -GO 3,5% 2% 5% 6% 3% 0-8%

Giy osylaties-G! 1.5% 2% i. > 2.5% 0.5% 0-4.5%

Glycosykti a- G2 ^r 0% 0%, 0% 0% ' 0% 0%

Glyco$ytatkti - GOF 45% 48% 5!% 44% 52% 44-34%

Glycoayiation-GiF 20% 22% 18% 16% 20% 35-24%

Glycosylate*!- G2F 4% 3% 5% 45% 6% 2-S%

Giycosyladon- 15% 1.8% !.?% 1.6% s.9% 0.5-3.9% Msa»o$« 5

Giycosylad a- 0% 0% 0% 0% 0% 0% Maano.se 8

C-terixusai lysine 0.5% .S% 1% 1.4% \3% 0-3.3% content* 2 iy iaes

C-terminal lysine 5% 4% 3% 2% 4% 2-?% consent- Ϊ hj c

eamidanon 3% 3.5% 3.2% 4% 3.5% 2-6%

Oxidation 2% 2.5% 2 ;% 1,8% 3% 0.8-5%

Aggregation 0.5% 0.4% 0.5% 0.4% 0.3% 0>2.S%

E AMPLE 2 A RECIPE FOR BIOSIMILAR OF BERCEPTIN& WITH A SIMILAR GLYCOSYLATiON

[0070] This example demonstrates one method to obtain a recipe for making a biosiraifar of Herceptin© focusing on optimization of the glyeosy!ation pattern.

Herceptin® (IN :Tra uzumab) is a humanized monoclonal antibody directed against the externa; doma n of the human HER2. The antibody is an IgG L consisting of wo y _{ heavy chains, two κ chains, and a single complex-type biantennary N-linked giycan at Asn300 of the heavy chain. For the purpose of this example Herceptin® (INN: rastuzumab) is a reference product. Five different batches of Hereeptin® were analyzed for glycOsylation pattern using 2AB giycan labeling method and the results are shown in Table 2, Since the modification identity for some chromatography peaks remains unknown, not all peaks could he assigned to specific modifications. Therefore, modifications have been labeled using peak numbers. An example of a chromatogram showing the giycan peaks representing different modifications from the 2AB giycan method with labeled peaks is shown in .Figure 3. To set target profile, the measurements for each modification for 5 batches of Kerceptint- were first collected. For example for Peak 1 modification me range was shown to be 1.7-2.8%. Based on the method shown in Example 1 , the target profile was identified to be 0.7-4.8% (lower limit was extended by 1 % and upper limit was extended by 2%).

Table 2 Setting Targe Profile For Glycaa Species OH I!ereeptin®

[Peak 9 (1 _S 3)G1 F If 0.3 10,6 .10.4 10.6 9.M 2.6%

[Peak 10 G2F 5.6 4,9 5.2 6.1 3.4-8, 1 %

Ipeak Π .9 ai 1 ,0 1.2 0.6 0-3.2%

Peak 12 fcj 0.4 0.4 0.4 0.3 0-2.4%

Peak 13 0.3 0.4 0.4 0.4 0.4 0-2,4%

Peak 1 .7 0.8 0,8 0,9 LO 0-3.0%

Peak 1 5 |0.3 0.5 0.5 0.5 0.7 0-2.7%

[0071 ] To obtain a recipe for production of a biosimilar with a similar glycosy!ation pattern to the original. Hercep n^, ("HO cells engineered to express the recombinant protein with an amino acid sequence identical to irastusju ab were first grown in the growth media without any inhibitors to establish a baseline. The gj ean species were analyzed using 2AB g!ycan method. The daia generated for the Baseline is shown in Table 3. It was observed that Peak 2 (GO) and Peak 6 (01 ), and Peak 7

(raannose-5 and GV) modifications were lower for the biosirnilar than their target profile.

[0072] GO, 01 and G1 ' modifications are non.~fueosyj.ated modifications and are controlled by a host protein called fucosyl transferase and the mannose~5 modification is controlled by the host protein known as a-mannoski&se L Fucosyl transferase can be inhibited by a variety of fucosy!transferase inhibitors shown in Figure 2, a-mannosidase 1 can be inhibited by kifunensine.

[0073] The result of optimization is shown in Method 1 in Table 3. Briefly to obtain trastuzu ab with modifications in the target range, ceils were placed in growth media and treated with 2F-Peraeetyl~Fucose (F'H) on day 7 at different concentrations (20μ , ΊΟμΜ, 5μΜ, Ι Μ, Ο. ΙμΜ) to identify optimal drug concentration. On day 1 2 cells were harvested and the trastuzuraab hiosin !ar isolated. 2AB glycan analysis of the bioshnilar showed that while 20μΜ FT! treatment resulted in an increase of GO. O l and f PTMs above thai of target PTMs, 10μΜ FTI treatment resulted in GO, Gl and Gl ^* levels thai matched the target PTM range. When cells were tr ated with FTI at concentrations lower than 8μΜ the modification were outside the target range. FTI concentrations used to reach target profile are cell specific so it is expected that different concentrations of the FTI or other modulators xvookl be required when a starting cell line is different from the one described in this example,

[0074] Different treatment methods such as Method 2 can be used to obtain target profile. For example, FTI can be added on a daily basis starting on day 5 (Table 3, Method 2} rather than on Day 7. Treatment of ceils expressing trastuzumab biosim lar with FTI at about .1.5-3.5μ everyday starting on Day 5 produced similar results to the one time treatment on Day 7 described in Method I . Based on these results, different treatment schedules of FTI (different methods) can be employed to obtain the same effect.

[0075] in addition to demonstrating that fucosyl transferase activity can be modulated, this Example also demonstrates modulation of the activity of a-mannosidase I using kifunensine in Method 3. Method 3 demonstrates optimization of the mannose species by addition of kifunensine. Different amounts of kifunensine (KFI) were added on day 7 ranging from about 0.001 ng ml - 100 ng/mi. The ideal concentration was identified as being between about 1 -10 ng/ml treated on Day 7. Since mannose-5 modification is not an important contributor to the biological activity of trastuzumab, this modulator may, but doesn't have to be included, in the recipe depending on the growth media used.

Table 3 Methods for Modulating modifications on a lYasio¾unmb Biosimilar

EXAMPLE 3 Determining Recombinant Protein Variants A.nd Their Biological Activity

'0()?6 j This example describes a method for determining recombinant protein variants and their ^' biological activity. [0077] The difference between product, modification and product variant is that product modifications can be measured and product variants cannot.. A. single or several product mod fications can be measured at the same time depending on the analytical method used. n the example below, there are two modifications on a recombinant protein product, modification 1 and 2. There are also other measurements thai were made thai provide additional information about the product, such as that 25% of the product is not modified as well as that 25% of the product contains two modifications. Based on this information, one skilled in the art can determine that the product, is a complex mixture of 4 product variants; product variant # 1. contains 2 modifications and is present at 25% in a complex mixture, product variant #2, containing only modification 1 , is present in the complex mixture at the abundance of 25%, product variant 3 is present at 25% and unmodified nroduct variant # 4 is also present at 25%.

[0078] Furthermore, the set of modifications on product variant n\ is modification 1 and 2, the set of modifications on product variant 2 is only one

modification #1, the set of modifications on product variant # 3 is modification 2; product variant 4 has no modifications.

[00791 The rationale or determining the type and the abundance of product variants aud not modifications ^' because it is the product variants, and not product modifications that exert the biological activity. The biological activity of the complex mixture is the sum of bioiouieal activities of each variant.

Product Variants

od ficatio s Abunda ce of

Product Variants

Abundance 1 :2.5$·

Abundance 2:255,

Abundance 3;2B¾

Abundance :25