CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application 63/320,936 filed on March 17, 2022 the entire contents of which is hereby incorporated by reference.

BACKGROUND

[0002] Methods that can effectively separate molecules or particles of varying size for analysis can be a valuable tool in drug development. For protein-based therapies, determination of fractions of monomeric versus aggregated species is essential (Roberts 2014), as aggregation can potentially affect drug activity, immunogenicity, and pharmacokinetic and pharmacodynamic profiles (Rambaldi et al. 2011). For small molecule formulations, particle size and shape can influence physical properties of the product, including dissolution rate and bioavailability of the active ingredients, drug release rate for modified-release formulations, content and uniformity, and flow and packing properties (Shekunov et al. 2006).

[0003] Field flow fractionation (FFF) is a family of methods for separating molecules or particles by hydrodynamic size The methods involve injecting the molecules in a liquid medium through a thin channel, and applying a cross-force that is perpendicular to the flow of the medium, which concentrates the molecule towards a porous membrane. The perpendicular force may be applied as a flow of liquid (Giddings 1973), or may be applied as another force such as electrical, gravitational, or thermal field (Giddings 1993). Smaller particles diffuse away more quickly from the porous membrane and elute earlier from the channel, while larger particles are more susceptible to the cross-force. Once the molecules examples exit the channel, they pass one or more detectors (for example, absorbance, light scattering, etc.') for quantitation or other analyses.

[0004] While FFF is an effective tool for separating molecules, current techniques can be associated with low recovery. Thus, a need for improved methods of performing FFF remain in the art. SUMMARY

[0005] The present disclosure recognizes certain challenges with the separation of molecules, e.g., in the development of biopharmaceutical drug products. For example, the present disclosure recognizes that the use of size exclusion chromatography (SEC) is limited by particle size. This is particularly challenging when separating biomolecules such as viral vectors, e.g., adeno- associated viral vectors (AAVs), nanoparticles, bacteria, viral particles, or exosomes, which can have sizes that are near the upper limit of SEC separation.

[0006] Among other things, the present disclosure provides technologies that can address certain limitations identified in existing methods for separating molecules, e.g., in the development of drug products. The technologies provided herein are particularly useful for separating molecules of interest (e g., a viral vector) from impurities or aggregates. In some embodiments, field flow fractionation (FFF) is useful for separating molecules (e.g., viral vectors, nanoparticles, bacteria, viral particles, or exosomes) because there is no upper size limit in particles that can be separated with FFF. Also provided herein are technologies such as crossflow gradients which can further improve FFF separation. In some embodiments, increasing a crossflow gradient can increase the resolution of FFF separation. In some embodiments, methods disclosed herein can increase the resolution of separation between a molecule of interest (e.g., a viral vector) and an impurity.

[0007] Some embodiments of the present disclosure are disclosed below. Additional embodiments are described in the Detailed Description, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present disclosure.

[0008] In some embodiments, the disclosure provides a method of separating particles in sample by FFF. In some embodiments, the disclosure provides a method of increasing resolution of separating particles of varying size in a sample by FFF. In some embodiments, the disclosure provides a method of purifying a sample by FFF, in which the sample comprises an active agent and one or more impurities.

[0009] The methods of the present disclosure comprise injecting the sample into a mobile phase that flows through a channel in a first direction that is parallel to the channel; and applying a force in the channel in a second direction that is transverse to the first direction (a “cross-force”), to separate the particles, active agent, and/or one or more impurities before they leave the channel. The application of the cross-force comprises at least three time periods: (i) a first time period, comprising applying the cross-force at a first magnitude; (ii) a second time period, comprising applying the cross-force at an increasing magnitude to a maximum magnitude that is greater than the first magnitude; and (iii) a third time period comprising applying the cross-force at a decreasing magnitude to a minimum magnitude that is less than the first magnitude.

[0010] In some embodiments, the cross-force comprises a flow of liquid, such as a flow of the mobile force; an electrical field; a gravitational field; or a thermal field.

[0011] In some embodiments, the maximum magnitude is about 25% greater than the first magnitude. In some embodiments, the maximum magnitude is about 50% greater, or about 100% greater, than the first magnitude.

[0012] In some embodiments, the maximum magnitude is at least 25% greater than the first magnitude. In some embodiments, the maximum magnitude is at least 50% greater, or at least 100% greater, than the first magnitude.

[0013] In some embodiments, the minimum magnitude is about 25% less than the first magnitude. In some embodiments, the minimum magnitude is about 50% less, or about 75% less, than the first magnitude.

[0014] In some embodiments, the minimum magnitude is at least 25% less than the first magnitude. In some embodiments, the minimum magnitude is at least 50% less, or at least 75% less, than the first magnitude.

[0015] In some embodiments, the application of the cross-force comprises a fourth time period, which comprises applying the cross-force at the minimum magnitude for a duration of time.

[0016] In some embodiments, the methods may further comprise collecting the separated particles after the particles leave the channel; or further comprise collecting the active agent, one or more impurities, or combination thereof, after the active agent, one or more impurities, or combination thereof leaves the channel.

[0017] In some embodiments, the methods may further comprise analyzing the separated particles after the particles leave the channel; or further comprise analyzing the active agent, one or more impurities, or combination thereof, after the active agent, one or more impurities, or combination thereof leaves the channel. In some embodiments, the analysis is by electron microscopy, scan cytometry, photon correlation spectroscopy, multiangle light scattering, Fourier-transform infrared spectroscopy (FT-IR), inductively coupled plasma mass spectrometry (ICP-MS), or a combination thereof.

[0018] In some embodiments, the channel comprises a flat channel. In some embodiments, the channel comprises a tubular channel. In some embodiments, the channel comprises a hollowfiber channel.

[0019] In some embodiments, the particles or active agent comprise protein aggregates, viral vectors, exosomes, mRNA lipid nanoparticles, bacteria, or viral particles.

BRIEF DESCRIPTION OF THE DRAWING

[0020] The figures described below, which together make up the Drawing, are for illustration purposes only, and are not intended to limit the disclosure.

[0021] FIGS. 1A-1B show the standard (#1) and test (#2-6) gradient profiles of the crossflows applied to a monoclonal antibody sample injected into the asymmetric flow FFF system described in Example 1 (FIG. 1A), and a fractogram illustrating the separation between the monomer peak and high molecular weight (HMW) peak resulting from the crossflow of each gradient profile (FIG. IB).

[0022] FIGS. 2A-2B show the standard (1) and test (2) gradient profiles of the crossflows applied to a monoclonal antibody sample injected into the asymmetric flow FFF system described in Example 2 (FIG. 2A), and a fractogram illustrating the separation between the monomer peak and the HMW 1 and HMW2 peaks generated from the crossflow of each gradient profile (FIG. 2B).

[0023] FIGS. 3A-3B show the standard (1) and test (2) gradient profiles of the crossflows applied to an adeno-associated virus (AAV) sample injected into the asymmetric flow FFF system described in Example 5 (FIG. 3A), and a fractogram illustrating the separation between the monomer peak and the HMW 1 and HMW2 peaks generated from the crossflow of each gradient profile (FIG. 3B). [0024] FIGS. 4A-4B show the standard (1) and test (2) gradient profiles of the crossflows applied to a monoclonal antibody sample injected into the hollow-fiber flow FFF system described in Example 6 (FIG. 4A), and a fractogram illustrating the separation between the monomer peak and the HMW peak generated from the crossflow of each gradient profile (FIG.

4B)

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0025] In order that the present disclosure can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. 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 disclosure is related.

[0026] Any headings provided herein are not limitations of the various embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0027] All of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present disclosure. Documents incorporated by reference into this text are not admitted to be prior art.

Definitions

[0028] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0029] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). [0030] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0031] Furthermore, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” is intended to include A and B, A or B, A (alone), and B (alone).

Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0032] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and/or “consisting essentially of’ are included.

[0033] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value encompassed by the range. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10.

[0034] A second number that is a “percent greater” than a first number is determined by multiplying the first number by the percentage, and then adding that product to the first number. For example, 15 is 50% greater than 10 (because 15 = (50% x 10) + 10), 180 is 200% greater than 60 (because 180 = (200% x 60) + 60), etc.

[0035] A second number that is a “percent less” than a first number is determined by multiplying the first number by the percentage, and then subtracting that product from the first number. For example, 15 is 50% less than 30 (because 15 = 30 - (50% x 30)), 48 is 20% less than 60 (because 48 = 60 - (20% x 60)), etc.

[0036] An “active agent” is an agent which itself has biological activity, or which is a precursor or prodrug that is converted in the body to an agent having biological activity. Active agents useful in the methods of the disclosure include, for example, protein aggregates, viral vectors (e.g., AAV), exosomes, mRNA lipid nanoparticles, bacteria, or viral particles.

[0037] A “standard” cross-force gradient profile is a profde in which the cross-force is either maintained at a constant magnitude, or is applied at an initial magnitude and decreased to a lower magnitude.

[0038] The term “resolution” as used herein refers to the separation of at least two components in a mixture, e.g., a sample. In some embodiments, the separation may be of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, or more components. The calculation of resolution may be in accordance with the U.S. Pharmacopeia guidelines (USP 2007).

Methods

[0039] The present disclosure provides an improved method of performing FFF that enhances separation of particles in a sample. In a typical FFF run, the cross-force is either constant or begins as constant and then decreases (Giddings 1993; Marioli & Kok 2019). The cross-force applied in analytical flow field flow fractionation (AF4) and hollow fiber flow field flow fractionation (HF5) is perpendicular to the flow of a stream of liquid flowing through a channel and is another flow referred to as a crossflow. The inventors have unexpectedly discovered that increasing the magnitude of the cross force (e.g., after elution of one peak) before decreasing to a final force, can lengthen retention time of the particles and improve their separation.

[0040] The methods of the present disclosure comprise injecting a sample comprising particles into a mobile phase that flows through a channel; and applying a cross-force in the channel that is traverse, or perpendicular, to the flow of the mobile phase. In some embodiments, application of a force comprises at least three time periods: (i) a first time period that comprises applying a force at a first magnitude; (ii) a second time period that comprises applying a force at an increasing magnitude to a maximum magnitude that is greater than a first magnitude; and (iii) a third time period that comprises applying a force at a decreasing magnitude to a minimum magnitude that is less than a first magnitude. In some embodiments, force can be applied at one or more additional time periods. [0041] In some embodiments, a duration (e.g., a total duration) comprises one or more time periods in which a force, e.g., a same or different force, is applied. In some embodiments, a duration (e.g., a total duration) comprises 1, 2, 3, 4, 5, or more time periods in which a force is applied. In some embodiments, a duration (e.g., a total duration) comprises 3 time periods, e.g., a first time period, a second time period, and a third time period, in which a force is applied.

[0042] In some embodiments, a first time period comprises no more than about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% of a total duration that a cross-force is applied

[0043] In some embodiments, a first time period comprises at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of a total duration that a cross-force is applied.

[0044] In some embodiments, a first time period may comprise about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, or about 10% to 40%, of a total duration that a cross-force is applied.

[0045] In some embodiments, a second time period comprises no more than about 40%, about 30%, about 20%, about 10%, or about 5% of a total duration that a cross-force is applied.

[0046] In some embodiments, a second time period comprises about 2% to about 40%, about 5% to about 40%, about 10% to about 40%, about 15% to about 40%, about 20% to about 40%, about 30% to about 40%, or about 5% to 30%, of a total duration that a cross-force is applied.

[0047] In some embodiments, a second time period comprises at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of a total duration that a cross-force is applied.

[0048] In some embodiments, a third time period comprises no more than about 40%, about 30%, about 20%, about 10%, or about 5% of a total duration that a cross-force is applied.

[0049] In some embodiments, a third time period comprises about 2% to about 40%, about 5% to about 40%, about 10% to about 40%, about 15% to about 40%, about 20% to about 40%, about 30% to about 40%, about 5% to about 30%, or about 5% to about 25%, of a total duration that a cross-force is applied.

[0050] In some embodiments, a third time period comprises at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of a total duration that a cross-force is applied. [0051] In some embodiments, a maximum magnitude of a cross-force may be about 10% greater than a first magnitude. In some embodiments, a maximum magnitude may be about 25%, or about 50%, or about 75%, or about 100%, or about 125%, or about 150%, or about 200% greater, than a first magnitude. In some embodiments, a maximum magnitude may be about 10% to 300%, or about 20% to 200%, or about 50% to 200%, greater than a first magnitude.

[0052] In some embodiments, a maximum magnitude of a cross-force may be at least 10% greater than a first magnitude. In some embodiments, a maximum magnitude may be at least 25%, at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 200% greater, than a first magnitude.

[0053] In some embodiments of the disclosure, an increase from a first magnitude of a crossforce to a maximum magnitude may be at a constant rate, e g., the amount that the force is increasing per unit time is the same throughout the duration of the second time period. In some embodiments, an increase from a first magnitude of a cross-force to a maximum magnitude is not at a constant rate; for example, the increase may accelerate or decelerate during the second time period. In some embodiments, during an increase from a first magnitude of a cross-force to a maximum magnitude, there may be one or more durations in which a magnitude of a cross-force is constant before continuing to increase towards a maximum magnitude.

[0054] In some embodiments, a minimum magnitude may be about 10% less than a first magnitude. In some embodiments, a maximum magnitude may be about 20%, or about 40%, or about 60%, or about 80%, less than a first magnitude. In some embodiments, a maximum magnitude may be about 10% to about 99%, or about 20% to about 99%, or about 30% to about 95%, less than a first magnitude.

[0055] In some embodiments, a minimum magnitude may be at least 10% less than a first magnitude. In some embodiments, a maximum magnitude may be at least 20%, or at least 40%, or at least 60%, or at least 80%, less than a first magnitude.

[0056] In some embodiments of the disclosure, a decrease from a maximum magnitude of a cross-force to a minimum magnitude may be at a constant rate. In some embodiments, a decrease from a maximum magnitude of a cross-force to a minimum magnitude is not at a constant rate. In some embodiments, during a decrease from a maximum magnitude of a cross- force to a minimum magnitude, there may be one or more durations in which a magnitude of a cross-force is constant before continuing to decrease towards a minimum magnitude.

[0057] In some embodiments, application of a cross-force comprises a fourth time period. In some embodiments, a fourth time period comprises applying a force at a minimum magnitude.

[0058] A channel disclosed herein may be any suitable channel known in the art for use with FFF. For example, the channel may comprise an entry port through which the mobile phase enters the channel, in which the port is located at or near a first end of the channel; an injection port through which the sample is injected into the mobile phase; and an exit port through which the active agent, impurities, or a combination thereof, of the sample leave the channel. The channel may further comprise one or more detectors (e.g., an absorbance detector, a lightscattering detector, etc.) positioned near or at the exit port. In some embodiments, the one or more detectors may be positioned near the exit port but outside of the channel.

[0059] In some embodiments of the disclosure, a cross-force may comprise a flow of liquid. The liquid may be the same as the mobile phase that flows through the channel. In some embodiments, the channel may be suitable for use in symmetrical flow FFF, in which the crossflow enters through a porous frit (typically at the top of the channel) and exits through a semi- permeable membrane outlet frit on the opposite side (typically the bottom of the channel). In some embodiments, the channel may be suitable for asymmetric flow FFF, in which only one semi-permeable membrane is located on the side of the direction of the cross-flow. In some embodiments, the channel is suitable for hollow fiber flow FFF, in which the channel typically comprises a cylindrical polymeric hollow-fiber membrane.

[0060] In some embodiments, a cross-force may be generated by a thermal field, for example, by applying a temperature gradient between a heated wall and a cooled opposite wall. In some embodiments, a cross-force may be generated by an electrical field, for instance, by applying a transverse electrical client. In some embodiments, a cross-force is generated by a gravitational field.

[0061] In some embodiments of the disclosure, the particles may comprise an active agent as described herein, e.g., protein aggregates, viral vectors (e.g., AAV), exosomes, mRNA lipid nanoparticles, bacteria, or viral particles. In some embodiments, particles may also comprise one or more impurities of an active agent. In some embodiments, particles may comprise a small molecule formulation, for example, a modified-release formulation (e.g., sustained release formulation, delayed-release formulation, etc.). In some embodiments, particles disclosed herein may comprise cells.

Exemplary Applications of Methods of the Disclosure

[0062] The methods of the present disclosure may be used to separate particles of varying size in a sample. Following separation, the particles may be analyzed, for example, to evaluate the size distribution or shape of the particles, or determine other physical characteristics of the particles. In some embodiments, the separated particles may be analyzed by, for example, electron microscopy, scan cytometry, photon correlation spectroscopy, multiangle light scattering, Fourier-transform infrared spectroscopy (FT-IR), inductively coupled plasma mass spectrometry (ICP-MS), or any combination thereof.

[0063] The methods of the present disclosure may also be used to increase resolution of FFF. In some embodiments, the increase in resolution may be assessed in accordance with the USP guidelines (USP 2007). In some embodiments, resolution may be increased by about 20%, or about 30%, or about 40%, or about 50%, as compared to the resolution determined from FFF applying a cross-force having a standard gradient profde. In some embodiments, resolution may be increased by at least 20%, or at least 30%, or at least 40%, or at least 50%, as compared to the resolution determined from FFF applying a cross-force having a standard gradient profde.

[0064] Further as disclosed herein, the methods of the present disclosure may be used to purify a sample that comprises, for example, an active agent. In some embodiments, the methods may further comprise collecting a separated active agent and/or disposal of non-active agent particles.

[0065] Also provided herein is a composition made according to any of the methods disclosed herein.

[0066] In some embodiments, a composition comprises an active agent, e.g., as disclosed herein. In some embodiments, an active agent comprises protein aggregates, antibodies, viral vectors (e g., AAV), exosomes, mRNA lipid nanoparticles, bacteria, viral particles, or combinations thereof.

[0067] In some embodiments, a composition is a pharmaceutical composition. [0068] In some embodiments, a composition produced using methods disclosed herein has less than 5-50% impurities, e.g., as compared to a similar composition made using a different method.

EXAMPLES

[0069] Studies were conducted to demonstrate that the resolution of FFF can be increased by applying the cross-force in accordance with methods of the disclosure.

Example 1

[0070] The impact on FFF resolution of including an increase in the magnitude of the cross force in accordance with methods of the present disclosure was studied using a flow FFF system that included a syringe-pump module for applying cross-flow. The system was equipped with a photodiode array, a multi-angle light scattering detector, and a refractive index detector. An asymmetric flow FFF channel was used that contained a 10 kDa polyethersulfone membrane.

[0071] A monoclonal antibody sample (10 pL at 5 mg/mL) was injected into the channel at a flow rate of 0.2 mL/min. Crossflows of six different gradient profiles as depicted in FIG. 1A, were applied. Each crossflow began at a flow rate of 1.5 mL/min and either remained at 1.5 mL/min (“standard” crossflow gradient profile, labeled as Gradient 1) or increased to a maximum flow rate of 2 mL/min, 2.5 mL/min, 3 mL/min, 3.5 mL/min, or 4 mL/min (“test” crossflow gradient profiles, labeled as Gradients 2-6, respectively). The increase in the flow rates was designed so that the monomer peak does not change shape. Each crossflow was decreased to a final flow rate of 0.5 mL/min after 19 minutes.

[0072] As shown in FIG. IB, the monomer peak eluted at about 12.5 minutes for all six crossflows studied. The high molecular weight (BMW) peak shifted to longer retention times as the crossflow maximum increased. In addition, the resolution as determined by U.S.

Pharmacopeia guidelines (USP 2007) increased from 0.79 to 2.46 as the change in crossflow increased from 0 to 2.5 mL/min (see Table 1). These results showed that application of a cross force in which the gradient profile included an increase in the magnitude of the force can improve the resolution of FFF. Table 1. Resolution calculated for each cross flow gradient.

„ ,. Crossflow Maximum USP Resolution

^Grad,en,s (mL/min) (HH)

1 (Standard) 1.5 0.79 ± 0.02

2 (Test) 2.0 1.05 ± 0.03

3 (Test) 2.5 1.53 ± 0.05

4 (Test) 3.0 1.73 ± 0.03

5 (Test) 3.5 2.05 ± 0.04

6 (Test) 4.0 2.46 ± 0.04

Example 2

[0073] The test crossflow gradient profile of the crossflows studied in Example 1 was modified to include a linear increase in the flow rate of the crossflow, followed by a decrease to a final flow rate lower than in Example 1. The FFF resolution resulting from this modified test gradient profile, which is in accordance with methods of the present disclosure, was compared to a standard gradient profile.

[0074] This study used the asymmetric flow FFF system described in Example 1. A monoclonal antibody sample (10 l. at 5 mg/mL) was injected into the channel at a flow rate of 0.2 mL/min was used in this study. The test crossflow gradient profile (labeled as Gradient 2) comprised a linear increase in flow rate from 1.8 mL/min to 3 mL/min followed by a linear decrease to a final flow rate of 0.1 mL/min, while the standard crossflow gradient profile (labeled as Gradient 1) comprised a flow rate of 1.8 mL/min that linearly decreased to a final flow rate of 0.1 mL/min (see FIG. 2A).

[0075] As shown in FIG. 2B, the application of crossflows in accordance with the test gradient profile and standard gradient profile both generated fractograms that exhibited a first HMW peak (HMW1) and a second HMW peak (HMW2); however, application of the crossflow according to the test gradient profile produced a greater separation between the monomer and HMW 1. The resolution between the monomer and HMW1, and between HMW1 and HMW2, for both cross flow gradient profiles is summarized in Table 2. Table 2. Resolution between peaks shown in fractograms generated by standard and test crossflow gradient profiles.

Standard Gradient Profile Test Gradient Profile

US ^p Resolution (HH) 0.71 ± 0.01 1.05 ± 0.02

(Monomer and HMWI)

USP Resolution (HH) 1.07 ± 0.02 1.09 ± 0.02

(HMW I and HMW2)

Example 3

[0076] The reproducibility of the results of Example 2 for the application of a crossflow in accordance with the test gradient profile was assessed by running the monoclonal sample on three different days, by two different operators, using two sets of membranes. The method showed good reproducibility overall. The results are summarized in Table 3.

Table 3. Results from assessing reproducibility of the study of Example 2 for application of a crossflow in accordance with the test gradient profile.

„ „ , _ _ , Number Monomer HMWI HMW2 Total

Day Operator Membrane _ ,

^J _{n/ n/} Samples % % % HMW%

Day l 1 1 8 86.4 ± 0.2 7.9 ± 0.1 5.6 ± 0.2 13.5 ± 0.2

Day 2 2 1 12 86.4 ± 0.3 7.3 ± 0.1 6.3 ± 0.2 13.5 ± 0.3

Day 3 2 2 12 86.5 ± 0.3 7.0 ± 0.3 6.4 ± 0.4 13.4 ± 0.3

All days (p ± o) 86.5 ± 0.3 7.3 ± 0.4 6.2 ± 0.4 13.5 ± 0.3

All days 100*(o/p) 0.3 5.5 6.6 2.0

Example 4

[0077] Fractions of the monomer, HMWI, and HMW2 of the monoclonal antibody sample determined by using asymmetrical flow FFF that applied a crossflow in accordance with the test gradient profile described in Example 2 were compared to fractions of the monoclonal antibody sample determined using size exclusion chromatography (SEC). As shown in Table 4, the results from each methodology were similar. Table 4. Comparison of fraction results determined by using asymmetrical flow FFF that applied a crossflow in accordance with the test gradient profde, and determined by using SEC.

Species Asymmetrical Flow FFF SEC

Monomer 86.5 87.7

HMW1 7.3 7.5

HMW2 6.2 4.6

Total HMW 13.5 12.2

Example 5

[0078] Asymmetrical flow FFF applying a crossflow in accordance with the methods of the present disclosure was evaluated using an adeno associated virus (AAV) sample. The results were compared to those from applying a crossflow having a standard gradient profile.

[0079] The crossflow comprised a test gradient profile (labeled as Gradient 2) comprising a linear increase in flow rate from 1.8 mL/min to 3 mL/min followed by a linear decrease to a final flow rate of 0.1 mL/min. The standard crossflow gradient profile (labeled as Gradient 1) comprised a flow rate of 1.8 mL/min that linearly decreased to a final flow rate of 0.1 mL/min (see FIG. 3A).

[0080] As shown in FIG. 3B, application of a crossflow having standard gradient profile produced a fractogram in which the HMW 1 peak appeared as a shoulder on the monomer peak. In contrast, the fractogram generated by applying the crossflow using the test gradient profile showed a clear separation between the monomer and HMW 1 peaks.

[0081] These results that used an AAV sample demonstrated that the improved results stemming from application of a cross force having the test gradient profile can occur across different therapeutic modalities. In some embodiments, methods disclosed herein are useful for separating an AAV particle from a sample comprising AAV particles and one or more additional components.

Example 6

[0082] The use of a cross-force that included an increase in cross-force magnitude in the gradient profile was demonstrated in a hollow-fiber force FFF system, in accordance with the methods of the present disclosure. The results were compared to those from applying a crossflow having a standard gradient profde.

[0083] The same flow FFF system of Example 1 was used, except that it was equipped with a hollow-fiber channel with a 10 kDa polyethersulfone membrane. A monoclonal antibody sample (10 pL at 5 mg/mL) was injected into the channel at a flow rate of 0.2 mL/min. The crossflow comprised a test gradient profile (labeled as Gradient 2) comprising a linear increase in flow rate from 0.4 mL/min to 0.7 mL/min followed by a linear decrease to a final flow rate of 0 mL/min. The standard crossflow gradient profile (labeled as Gradient 1) comprised a flow rate of 0.4 mL/min that linearly decreased to a final flow rate of 0 mL/min (see FIG. 4A).

[0084] As shown in FIG. 4B, the application of a crossflow according to the test gradient profile produced a greater separation between the monomer and HMW1, resulting in greater resolution between the peaks (see Table 5). The crossflow with either gradient profile did not result in two HMW peaks.

[0085] These results demonstrated that the improved results stemming from application of a cross force having the test gradient profile can occur in different FFF systems.

Table 5. Resolution between monomer and HMW peaks shown in fractograms generated by standard and test crossflow gradient profiles.

USP Resolution (HMM) Standard Gradient Profile Test Gradient Profile

Resolution 0.76 ± 0.01 1.36 ± 0.01

(Monomer and HMW)

REFERENCES FOR EXAMPLES 1-6

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