MODULATION OF CHARGE VARIANTS IN A MONOCLONAL ANTIBODY COMPOSITION

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/105,890, filed on January 21, 2015, the contents of which are incorporated by reference herein, in their entirety and for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically as a text file named Adalimumab_ST25.txt, created on January 19, 2016, with a size of 12,000 bytes. The Sequence Listing is incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates generally to the field of protein biochemistry. More

particularly, the invention relates to a purification scheme for monoclonal antibodies and other small binding peptides and proteins, which substantially reduces charge variants, thereby producing a more homogenous population of the antibody for patient

administration.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications, accession numbers, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety and for all purposes.

As part of the Biologies Price Competition and I nnovation Act (BPCIA), a biological drug product (produced in or derived from living organisms) may be demonstrated to be "biosimilar" if data show that, among other things, the product is "highly similar" to an already-approved biological product. The biosimilar product should retain at least the biologic function and treatment efficacy of the U.S. Food and Drug Agency-approved biological product.

Monoclonal antibodies (mAbs) may be used as therapeutic proteins. Purified monoclonal antibodies are most often present in a complex heterogeneous mixture based on chemical modifications of selected amino acids sites that range from subtle to significant. Understanding the impact of these modifications is of considerable importance in the biotechnology field. Monoclonal antibodies have charge heterogeneity that optimizes the balance of gaining favorable electrostatic interactions and determines their structure, stability, binding affinity, chemical properties and, hence, their biological activity.

Consistency of a biologic drug product, along with a maximized shelf life of the product are of paramount importance to drug developers and manufacturers. Short shelf life usually translates to manufacturing challenges and high costs of production by manufacturers.

During the cell culture or fermentation process, antibodies and proteins may undergo phenomena known as post-translational modifications. These modifications contribute to several forms of heterogeneity seen in therapeutic proteins. Additionally, there are forms of heterogeneity that occur during the manufacture caused by stresses imparted during the process such as size and charge that can occur due to enzymatic processes or spontaneous degradation and modifications. Monoclonal antibodies undergo chemical modification via several different mechanisms, including oxidation, deamidation, glycation, isomerization and fragmentation, that result in the formation of various charge variants and heterogeneity.

Chemical and enzymatic modifications such as deamidation, and sialylation, result in an increase in the net negative charge on mAbs and cause a decrease in pi values, thereby leading to formation of acidic variants. C-terminal lysine cleavage results in the loss of net positive charge leading to the formation of monoclonal antibody species with greater acidic charge. Another mechanism for generating acidic variants is the formation of various types of covalent adducts, e.g., glycation, where glucose or lactose can react with the primary amine of a lysine or arginine residue during manufacturing in glucose-rich culture media or during storage if a reducing sugar is present in the formulation. Formation of the basic variants can result from the presence of one or more C-terminal lysines or proline amidation, succinimide formation, amino acid oxidation or removal of sialic acid, which introduce additional positive charges or removal of negative charges; both types of modifications cause an increase in pi values.

Although there is substantial knowledge and experience with the degradation pathways that are active during production and formulation, a current challenge is to understand how the heterogeneity described above may affect efficacy, potency, immunogenicity and clearance. Little is known about the effects of charge on the PK of subcutaneously (SC) administered mAbs. Passage through the interstitium to the vascular or lymphatic capillaries can present a barrier to efficient drug absorption after SC

administration. Interstitial diffusion of mAbs is likely to be influenced by their charge and their electrostatic interactions with negatively charged constituents of the interstitial area underlying the dermis of the skin.

Recently, the growth and interest in the development of biosimilars has presented several unique challenges to the production of biotherapeutics such as mAbs. The development of innovative molecules allows latitude to define the product quality attributes (PQAs) and, ultimately, the critical quality attributes (CQAs) of a mAb during the natural course of the development process. This paradigm, in turn, permits the

implementation of a potentially robust production platform capable of handling the mAbs that a pipeline of candidates may produce with minimal optimization.

The development of biosimilar molecules, by contrast, imposes the confines of a predefined (by the innovator) set of product quality attribute ranges. The impact on process development is that the latitude that a platform process may afford may be significantly reduced by the requirement to fit within a defined range for multiple PQAs. This is especially true for those attributes that are known to be or could potentially be biologically relevant such as the charge variants described above. Purification or reduction of such heterogeneity so as to achieve a more homogenous population poses a significant challenge to process developers. The differences in the species that make up the heterogeneous population of charge variants are often quite subtle and similar in their characteristics to the primary mAb population of interest. Consequently, these unwanted variants are difficult to separate effectively while maintaining a reasonable mAb recovery. There is a need to minimize these unwanted variants toward a more homogenous population of biosimilar mAbs.

SUMMARY OF THE INVENTION

The disclosure features processes for removing both acid charge variants and basic charge variants from a monoclonal antibody preparation. In some aspects, the process comprises a succession of Protein A chromatography, followed by a mixed mode hybrid of anion exchange and hydrophobic interaction chromatography (AEX/HIC), followed by cation exchange chromatography (CEX). Orthogonal virus inactivation steps may be included in the process. The monoclonal antibody pool prepared according to this process, which includes non-variant antibodies and acid charge variants of such antibodies, may be concentrated and formulated for storage and patient administration.

In some detailed aspects, the process comprises loading a mammalian cell-expressed monoclonal antibody onto a support comprising Protein A having an antibody binding capacity of from about 10 g/L to about 60 g/L, and eluting the monoclonal antibody from the Protein A, thereby producing a first eluate comprising the monoclonal antibody;

optionally inactivating viruses in the first eluate, loading the first eluate onto an AEX/HIC support and allowing the first eluate to flow through the AEX/HIC support, thereby producing a flow-through pool comprising the monoclonal antibody, loading the flow- through pool comprising the monoclonal antibody onto a CEX support, monitoring the absorbance units and determining when the absorbance units, measured at UV A280, decrease about 7% to about 14% from the peak absorbance units, measured at UV A280, or about 7% to about 14% on a column volume basis, and then washing the CEX support with a wash buffer having a pH of from about 5.8 to about 6.6 and a conductivity target of from about 6.6 mS/cm to about 7.6 mS/cm for a period of time sufficient to remove a desired amount of acidic charge species of the antibody, and then eluting the monoclonal antibody from the CEX chromatography support in step with an elution buffer having a pH of from about 6.0 to about 6.4 and a conductivity target of from about 10 mS/cm to about 14 mS/cm, thereby producing a second eluate comprising the monoclonal antibody and from about 10% to about 20% by weight of acid charge variants of the monoclonal antibody. The percentage of acid charge variants may be determined according to any suitable technique known in the art. Non limiting examples of suitable techniques include imaged capillary isoelectric focusing, cation exchange chromatography, and high performance liquid chromatography.

To remove basic charge variants, the method may further comprise, before eluting the monoclonal antibody according, washing the CEX support with a buffer comprising a pH of from about 6.5 to about 7.0, and then eluting the monoclonal antibody from the CEX support. Following removal of basic charge variants, the second eluate comprises the monoclonal antibody, acid charge variants, and from about 15% to about 25% by weight of basic charge variants of the monoclonal antibody as determined by any suitable technique.

The process is preferably used in accordance with monoclonal antibodies that are expressed by mammalian expression host cells such as CHO or HEK293 cells. The antibodies preferably specifically bind to tumor necrosis factor alpha. The monoclonal antibodies may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2. The monoclonal antibodies are preferably adalimumab.

In some aspects, the Protein A support preferably is equilibrated with a buffer comprising about 20 mM sodium phosphate and about 50 mM sodium chloride, and a pH of from about 7 to about 7.4. In some aspects, the Protein A support is equilibrated with a buffer comprising about 20 mM sodium phosphate and about 50 mM sodium chloride, and a pH of from about 7.2. Equilibration precedes loading the expressed-monoclonal antibody preparation onto the support. Following loading and adsorption of monoclonal antibodies to the Protein A substrate, the support may be washed. Thereafter, the monoclonal antibodies may be eluted from the Protein A support with an elution buffer comprising about 80 mM acetic acid or sodium acetate and having a pH of about 3.5, thereby producing the first eluate comprising the monoclonal antibody. Following elution, the support may be re-equilibrated using the original equilibration buffer.

When included, the virus inactivation step may comprise adding citric acid to the eluate from the Protein A support until the pH of the Protein A eluate is from about 3.4 to about 3.6, and then holding the eluate at a temperature of from about 18° C to about 25° C for from about 50 to about 70 minutes and, following the holding, increasing the pH of the eluate to from about 7.3 to about 7.7 and then filtering the eluate. In some aspects, virus inactivation may comprise adding citric acid to the Protein A eluate until the pH of the eluate is about 3.5, and then holding the eluate at a temperature of from about 18° C to about 25° C for from about 50 to about 70 minutes and, following the holding, increasing the pH of the eluate to from about 7.4 to about 7.6 and then filtering the eluate. Filtering may comprise depth filtration followed by 0.2 micron filtration.

Following virus inactivation, if such inactivation is included, the filtered Protein A eluate (including the monoclonal antibodies as purified to this point) may be loaded onto a mixed mode chromatography support, for example, an AEX/HIC support. A non-limiting example of a suitable AEX/HIC support comprises a N-Benzyl-N-methyl ethanol amine ligand, which may have a binding capacity of from about 50 g/L to about 150 g/L. The support may be equilibrated with a buffer comprising about 50 mM HEPES and about 66 mM sodium chloride and having a pH of from about 7.4 to about 7.6. The loaded eluate is allowed to flow through the support, including the a N-Benzyl-N-methyl ethanol amine ligand. Following material loading, the support may be washed and re-equilibrated using the original equilibration buffer.

Following chromatography through the AEX/HIC support, the flow-through pool comprising the monoclonal antibody may be loaded onto a CEX support. A non-limiting example of a CEX support comprises a sulfopropyl or sulfonate ligand, which may have an antibody binding capacity of from about 25 g/L to about 65 g/L. Following loading and adsorption of monoclonal antibodies to the substrate, the CEX support may be washed, first with a buffer such as the equilibration buffer, and then with a washed with a second wash buffer having a higher conductivity to elute acid charge variants from the substrate, and then washed with a buffer such as the equilibration buffer in order to quench acidic charge elution. The second wash may be carried out for a duration of time sufficient to remove a desired amount of acidic charge species of the antibody. For example, for a biosimilar antibody, the desired amount may be the amount to achieve a level of acidic charge species in the CEX eluate that approximates the amount of acidic charge species in the reference antibody. Thus, in cases where a higher amount of acidic charge species are present in the flow-through pool from the AEX/HIC chromatography step, the duration of the second wash may be longer and, conversely, in cases where a lower of amount of acidic charge species are present in the flow-through pool from the AEX/HIC chromatography step, the duration of the second wash may be shorter.

In some aspects, acidic charge variants may be eluted from the CEX support by washing the support with a buffer having pH of from about 5.9 to about 6.5, or a pH of from about 6 to about 6.4, or a pH of from about 6.1 to about 6.3, or a pH of about 6.2 and/or a conductivity target of from about 6.8 mS/cm to about 7.4 mS/cm, from about 6.9 mS/cm to about 7.3 mS/cm, from about 7 mS/cm to about 7.2 mS/cm, or about 7.2 mS/cm. Such washing proceeds when the absorbance units, measured at UV A280, decrease from about 7% to about 14%, from about 8% to about 12%, from about 9% to about 11%, or about 10% from the peak absorbance units measured at UV A280, with such washing proceeding for a duration of time sufficient to remove a desired amount of acidic charge species of the antibody.

Following the wash to remove acidic charge species of the monoclonal antibody from the CEX support, the monoclonal antibody may be eluted from the CEX support. The antibody may be eluted by contacting the CEX support with an elution buffer that comprises about 100 mM sodium chloride, a pH of from about 6 to about 6.4, from about 6.1 to about 6.3, or about 6.2, and a conductivity target of from about 11 mS/cm to about 14 mS/cm, from about 12 mS/cm to about 14 mS/cm, from about 12 mS/cm to about 13 mS/cm, from about 12.3 mS/cm to about 13.3 mS/cm, from about 12.6 mS/cm to about 13 mS/cm, or about 12.8 mS/cm. The 100 mM sodium chloride may be attained by adding sodium chloride to the elution buffer over a gradient (e.g., increasing the concentration over time). The sodium chloride may be added to the elution buffer via a linear gradient or via a stepwise gradient.

The eluate from the CEX support comprises purified monoclonal antibodies, and from about 9% to about 15% by weight of acid charge variants of the monoclonal antibody. In some aspects, the eluate comprises from about 10% to about 20% by weight of acid charge variants of the monoclonal antibody, as determined by any suitable technique. In some aspects, the eluate comprises from about 11% to about 19% by weight of acid charge variants of the monoclonal antibody, as determined by any suitable technique. In some aspects, the eluate comprises from about 12% to about 19% by weight of acid charge variants of the monoclonal antibody, as determined by any suitable technique. In some aspects, the eluate comprises from about 14% to about 18% by weight of acid charge variants of the monoclonal antibody, as determined by any suitable technique. In some aspects, the eluate comprises from about 15% to about 17% by weight of acid charge variants of the monoclonal antibody, as determined by any suitable technique.

Optionally, the eluate from the CEX support, which comprises the monoclonal antibodies may be filtered. Optionally, the eluate from the CEX support, which comprises the monoclonal antibodies may be concentrated and/or diafiltered. Compositions comprising monoclonal antibodies and acidic and/or basic charge variants of the monoclonal antibodies prepared according to such methods are provided. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an overview of the purification process.

Figure 2 shows a protein uptake study design based on the MABSELECT SURE ^® resin. Figure 3 shows static binding capacity (g/L).

Figure 4 shows MabSelect SuRe ^® adsorption isotherm for adalimumab.

Figure 5 shows a relationship between dynamic binding capacity and residence time.

Figure 6 shows a relationship between dynamic binding capacity and linear velocity.

Figure 7 shows an experimental design for Protein A elution conditions.

Figure 8 shows MabSelect SuRe ^® elution study results.

Figure 9A and Figure 9B show contour maps showing the effect of pH and

conductivity on yield (Fig. 9A) and reduction of high molecular weight (Fig. 9B, shown in %change in HMW%) of CAPTO ^® Adhere for ada limumab.

Figure 10 shows contour maps showing the effect of pH and conductivity on acidic charge variants (A, left panel, shown in % change in acidic variants) and HCP (B, right panel) using CAPTO ^® Adhere for adalimumab.

Figure 11 shows a mixed mode acidic charge variant clearance and yield profile.

Figure 12 shows a mixed mode high molecular weight clearance and yield profile.

Figure 13A and Figure 13B show contour maps showing the effect of Wash 2 pH and conductivity on CAPTO ^® SP I mpRes eluate yield (Fig. 13A) and reduction of high molecular weight (Fig. 13B, shown in % change in HMW%) for adalimumab.

Figure 14 shows flux vs. TMP at initial adalimumab concentration (lOg/L).

Figure 15 shows flux vs. TMP at final adalimumab concentration (65g/L).

Figure 16 shows concentration factor vs. flux of adalimumab during concentration.

Figure 17 shows a scalable purification process (5L - 200L scale) for adalimumab.

Figure 18 shows the impact of mass load on yield and HMW using HEPES.

Figure 19 shows the impact of mass load on residual Protein A clearance using

HEPES.

Figure 20 shows the impact of mass load on HCP clearance using HEPES. Figure 21 shows the impact of mass load on acidic charge variant clearance using

HEPES.

Figure 22 shows the derivation of Wash-2 cutting criteria.

DETAILED DESCRIPTION OF THE INVENTION

Various terms relating to aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.

As used herein, the singular forms "a," "an," and "the" include plural referents unless expressly stated otherwise.

As used herein, the terms "comprising," "having," and "including" encompass the more restrictive terms "consisting essentially of" and "consisting of."

The terms subject and patient are used interchangeably, and include any animal. Subjects include mammals, including companion and farm mammals, as well as rodents, including mice, rabbits, and rats, and other rodents. Non-human primates preferred subjects. Human beings are highly preferred subjects.

It has been observed in accordance with the invention that substantial acidic charge variants could be removed from an isolated and purified population of mammalian cell- expressed monoclonal antibodies that bind to tumor necrosis factor (TNF)-alpha. The resulting population of monoclonal antibodies had enhanced homogeneity relative to the initial expression pool of antibodies, including less than about 15% by weight of acid charge variants. The purification process included an initial Protein A capture purification, followed by an intermediate chromatography step using a hybrid of anion exchange and hydrophobic interaction chromatography (AEX/HIC), followed by a polishing chromatography step using cation exchange chromatography (CEX). Notably, CEX is typically used as an intermediate chromatography step, with AEX/HIC typically used as a polishing chromatography step, such that the process inverted the customary antibody preparation protocol. The process also included two orthogonal steps for virus inactivation/removal, as well as concentration and diafiltration steps. An overview of the process is shown in Figure 1. Accordingly, the invention features methods for removing charge variants, preferably acid charge species but also including basic species, from a monoclonal antibody composition. Compositions of monoclonal antibodies with reduced charge variants, as prepared using such methods, are also featured. The purification process may find use, for example, in the preparation of biosimilar monoclonal antibodies.

Purification processes according to the disclosure are suitable for any monoclonal antibody. I n some preferred aspects, the antibody specifically binds to an epitope on TNF- alpha, and the epitope may be linear or conformational.

Preferably, the antibody is a full length monoclonal antibody, comprising both variable and constant regions. In some aspects, the antibody may comprise a derivative or fragment or portion of a full-length antibody that retains the antigen-binding specificity, and also preferably retains most or all of the affinity, of the full length antibody molecule. The antibody may comprise post-translational modifications (PTMs) or moieties, which may impact antibody activity or stability. The antibody may be methylated, acetylated, glycosylated, sulfated, phosphorylated, carboxylated, and/or amidated, and may comprise other moieties that are well known in the art.

In some preferred aspects, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ I D NO: 1. In some preferred aspects, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2. Preferably, the antibody comprises a heavy chain constant domain and/or a light chain constant domain. The heavy and light chain amino acid sequences of the antibody may comprise those of U.S. Pat. No. 6,090,382.

In highly preferred aspects, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ I D NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, an example of which is adalimumab. adalimumab comprises a molecular mass of about 148 kDa in glycosylated form, and about 145 kDa in de-glycosylated form, and comprises an isoelectric point of about 8.35, and an extinction coefficient (280 nm) of about 1.4 M ^_1 cm adalimumab comprises two kappa light chains di-sulfide-bonded to two gamma heavy chains, and comprises 1330 amino acids.

Common PTMs for adalimumab include N-glycosylation, C-terminal variants (e.g., cleavage of lysine, proline amidation), N-terminal pyro-E formation, oxidation,

isomerization, deamidation, succinimide formation, mannosylation, and glycation. Moieties include any chemical group or combinations of groups commonly found on immunoglobulin molecules in nature, or otherwise added to antibodies by recombinant expression systems, including prokaryotic and eukaryotic expression systems.

The production of monoclonal antibodies and other proteins preferably is carried out in host cells such as mammalian or bacterial systems. The monoclonal antibody can be located in different locations within the cell or in some cases secreted from the cell into the media. In the latter case, the cells can be removed and separated from the monoclonal antibody by using centrifugation and/or filtration. The antibody is preferably expressed using mammalian cells. Non-limiting examples of suitable mammalian expression hosts include Chinese Hamster Ovary (CHO) cells and human embryonic kidney 293 (HEK293) cells.

The recombinantly expressed antibody may be clarified from its mammalian host cells by either a two stage depth filtration or centrifugation process. The depth filters may comprise cellulose fiber and diatomaceous earth. Following depth filtration, the material may be passed through a 0.2 μιη filter to achieve the clarified cell culture supernatant. The clarified cell culture supernatant can then be purified using the process as described below.

Following expression, the preparation of monoclonal antibodies is purified in order to remove contaminants, including charge variants of the monoclonal antibodies

themselves. As a first step, the antibody preparation may be loaded onto a support comprising Protein A, whereby the antibodies interact with the Protein A. The support preferably comprises particles that may be packed into a chromatography column. The Protein A preferably has an antibody binding capacity of from about 10 g/L to about 100 g/L, more preferably from about 10 g/L to about 60 g/L, and more preferably from about 20 g/L to about 50 g/L. MABSELECT SURE ^® Protein A media is an example of a suitable Protein A support. UNOsphere SUPrA™ media, PROSEP ^® Ultra Plus Protein A media, and ABSOLUTe ^® High Cap Protein A media are other examples of suitable Protein A supports. Any suitable Protein A support available in the art may be used.

Prior to loading the antibody preparation onto the Protein A support, the support is preferably equilibrated. The equilibration medium may comprise a buffer. The buffer may comprise from about 5 mM to about 50 mM of sodium phosphate and from about 20 mM to about 80 mM of sodium chloride. The buffer may comprise from about 10 mM to about 30 mM of sodium phosphate, from about 12 mM to about 28 mM of sodium phosphate, from about 15 mM to about 25 mM of sodium phosphate, from about 17 mM to about 23 mM of sodium phosphate, from about 15 mM to about 20 mM of sodium phosphate, from about 20 mM to about 25 mM of sodium phosphate, from about 15 mM to about 30 mM of sodium phosphate, from about 18 mM to about 22 mM of sodium phosphate, from about 19 mM to about 21 mM of sodium phosphate, or about 20 mM of sodium phosphate. The buffer may comprise from about 30 mM to about 70 mM of sodium chloride, from about 35 to about 65 mM of sodium chloride, from about 40 mM to about 60 mM of sodium chloride, from about 42 mM to about 58 mM of sodium chloride, from about 45 to about 55 mM of sodium chloride, from about 40 mM to about 50 mM of sodium chloride, from about 50 to about 60 mM of sodium chloride, from about 48 to about 52 mM of sodium chloride, from about 49 mM to about 51 mM of sodium chloride, or about 50 mM of sodium chloride. In some detailed aspects, the buffer comprises about 20 mM of sodium phosphate and about 50 mM of sodium chloride. As an alternative to sodium phosphate, the buffer may comprise HEPES, Bis-Tris, MES, a succinate salt, or a citrate salt. As an alternative to sodium chloride, the buffer may comprise sodium sulfate or ammonium sulfate. The pH of the buffer may be from about 5 to about 9, preferably from about 6 to about 8, from about 6.8 to about 7.6, from about 6.9 to about 7.5, from about 7.0 to about 7.4, from about 7.1 to about 7.3, or about 7.2. The pH of the buffer may be about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, or about 7.6.

The conductivity target of the equilibration (mS/cm) may range from about 1 mS/cm to about 40 mS/cm. The conductivity target of the equilibration (mS/cm) is preferably from about 6 mS/cm to about 10 mS/cm. The conductivity target may be from about 6 mS/cm to about 8 mS/cm, from about 7 mS/cm to about 9 mS/cm, from about 7 mS/cm to about 10 mS/cm, from about 8 mS/cm to about 10 mS/cm, from about 8 mS/cm to about 9 mS/cm, or about 8 mS/cm.

Loading of the antibody preparation onto the Protein A support is carried out at a temperature, in a volume, and for a time suitable to allow for maximal adsorption of the monoclonal antibodies to the Protein A ligand. Undesired materials that do not adsorb to the Protein A ligand flow through the support during chromatography. To further remove undesired materials that adhere to the ligand or to the antibody protein, the antibody- adsorbed support is preferably washed. Any suitable number of washes may be used, and the wash may comprise a buffer and sufficient stringency to remove undesired materials but not elute antibodies from the Protein A. The wash medium preferably is the same as the equilibration medium, in terms of make-up (e.g., sodium phosphate and sodium chloride) and pH.

The conductivity target of the wash may range from about 1 mS/cm to about 50 mS/cm. The conductivity target of the wash is preferably from about 6 mS/cm to about 10 mS/cm. The conductivity target may be from about 6 mS/cm to about 8 mS/cm, from about 7 mS/cm to about 9 mS/cm, from about 7 mS/cm to about 10 mS/cm, from about 8 mS/cm to about 10 mS/cm, from about 8 mS/cm to about 9 mS/cm, or about 8 mS/cm.

Following the wash, the monoclonal antibodies are eluted from the Protein A support. Elution may be carried out at a temperature, in a volume, and for a time suitable to allow for maximal elution yield of the monoclonal antibodies from the Protein A ligand. Elution buffer is preferably acidic. The elution buffer may comprise from about 10 mM to about 300 mM, from about 40 mM to about 120 mM acetic acid or sodium acetate, from about 50 mM to about 110 mM acetic acid or sodium acetate, from about 60 mM to about 100 mM acetic acid or sodium acetate, from about 70 mM to about 90 mM acetic acid or sodium acetate, from about 72 mM to about 88 mM acetic acid or sodium acetate, from about 75 mM to about 85 mM acetic acid or sodium acetate, from about 78 mM to about 82 mM acetic acid or sodium acetate, from about 79 mM to about 81 mM acetic acid or sodium acetate, from about 80 mM to about 90 mM acetic acid or sodium acetate, from about 70 mM to about 80 mM acetic acid or sodium acetate, of about 80 mM acetic acid or sodium acetate. As an alternative to acetic acid or sodium acetate, the elution buffer may comprise sodium citrate, sodium formate, or glycine. Sodium acetate is preferred. The elution buffer may comprise a pH of from about 2 to about 4.5, from about 3 to about 4, from about 3.2 to about 3.8, from about 3.3 to about 3.7, from about 3.4 to about 3.6, from about 3.3 to about 3.5, from about 3.5 to about 3.7, about 3.3, about 3.4, about 3.5, about 3.6, of about 3.7. Elution of the monoclonal antibody produces an eluate comprising the monoclonal antibody.

The eluate comprising the monoclonal antibody may be treated to inactivate any viruses present in the eluate. The virus inactivation may comprise acidifying the eluate at a temperature and for a period of time sufficient to inactivate any viruses present in the eluate. The acidification may comprise, for example, adding acetic acid, citric acid, hydrochloric acid, formic acid, or combination thereof to the eluate until a desired pH is achieved. The resultant pH of the acidified eluate may be from about 3 to about 4, from about 3.2 to about 3.8, from about 3.3 to about 3.7, from about 3.4 to about 3.6, from about 3.3 to about 3.5, or from about 3.5 to about 3.7, or about 3.3, about 3.4, about 3.5, about 3.6, or about 3.7. The eluate may be warmed to a temperature of from about 4° C to about 25° C, and more preferably from about 18° C to about 25° C. The eluate may be warmed before, during, or after acidification. Once at the desired inactivation temperature, the eluate is preferably maintained at both the pH and temperature for a period of time sufficient to inactivate substantially all latent viruses in the eluate. The eluate may be so maintained for about 10 to about 90 minutes, preferably from about 50 to about 80 minutes, and more preferably from about 60 to about 70 minutes.

After this virus inactivation hold time elapses, the pH of the eluate may be increased, for example, by addition of a suitable basic buffer such as Tris. The resultant pH of the eluate may be from about 7.3 to about 7.7, from about 7.3 to about 7.6, from about 7.3 to about 7.5, from about 7.4 to about 7.7, from about 7.4 to about 7.6, from about 7.4 to about 7.5, or from about 7.5 to about 7.7 or about 7.3, about 7.4, about 7.5, about 7.6, or about 7.7. Following the pH increase, the eluate may be filtered, for example, with a sterilizing grade 0.22 μιη filter. Depth filtration may be used to filter the eluate, preferably as a first step of the filtration process. The depth filters may comprise mixed cellulose esters and borosilicate glass. Thus, depth filtration may precede sterilizing with the 0.22 μιη filter.

Following the virus inactivation step, or following the Protein A elution if virus inactivation is not included, the monoclonal antibody may be further purified with a second chromatography step. Preferably, the chromatography employed in this step comprises a hybrid of anion exchange and hydrophobic interaction chromatography (AEX/HIC). In some aspects, the chromatography media comprises a support comprising a N-Benzyl-N-methyl ethanol amine ligand. A non-limiting example of a suitable media includes CAPTO ^® Adhere media. In some aspects, the chromatography media comprises a support comprising a hexylamine ligand. In some aspects, the chromatography media comprises a support comprising a phenylpropylamine ligand. The ligands may be linked to any suitable support, including but not limited to an agarose support or a cross-linked cellulose support.

The eluate from the Protein A chromatography step, which may be the filtered eluate from the virus inactivation step, is preferably loaded onto an AEX/HIC

chromatography support. For example, an AEX/HIC chromatography support may comprise a N-Benzyl-N-methyl ethanol amine ligand (or hexylamine, phenylpropylamine, or other suitable ligand) and allowed to flow through the support. The support preferably has a binding capacity of from about 50 g/L to about 150 g/L, from about 60 g/L to about 140 g/L, from about 70 g/L to about 130 g/L, or from about 75 g/L to about 125 g/L.

Mass loading may play a role in the balance of purity versus yield. For adalimumab, from about 90 g/L to about 110 g/L of antibody may be loaded onto the AEX/HIC support. The mass loading of adalimumab may be from about 95 g/L to about 105 g/L, from about 96 g/L to about 104 g/L, from about 97 g/L to about 103 g/L, from about 98 g/L to about 102 g/L, from about 99 g/L to about 101 g/L, from about 95 g/L to about 100 g/L, from about 100 g/L to about 105 g/L, from about 90 g/L to about 105 g/L, from about 90 g/L to about 100 g/L, from about 100 g/L to about 110 g/L, from about 100 g/L to about 105 g/L, or about 100 g/L.

Prior to loading the eluate onto the AEX/HIC support, the support is preferably equilibrated. The equilibration medium may comprise a buffer. The buffer may comprise from about 10 mM to about 150 mM of HEPES, from about 25 mM to about 75 mM of HEPES, from about 30 mM to about 70 mM of HEPES, from about 35 mM to about 65 mM of HEPES, from about 40 mM to about 60 mM of HEPES, from about 45 mM to about 55 mM of HEPES, from about 47 mM to about 53 mM of HEPES, from about 49 mM to about 51 mM of HEPES, or about 50 mM of HEPES. In addition to HEPES, the buffer may comprise from about 40 mM to about 80 mM sodium chloride, from about 45 mM to about 75 mM sodium chloride, from about 50 mM to about 75 mM sodium chloride, from about 55 mM to about 75 mM sodium chloride, from about 55 mM to about 70 mM sodium chloride, from about 60 mM to about 70 mM sodium chloride, from about 64 mM to about 68 mM sodium chloride, from about 65 mM to about 70 mM sodium chloride, from about 65 mM to about 68 mM sodium chloride, from about 65 mM to about 67 mM sodium chloride, or about 66 mM sodium chloride. As an alternative to HEPES, the buffer may comprise sodium phosphate, potassium phosphate, Bis-Tris, MES, or a combination thereof. The pH of the buffer may be from about 6.0 to about 9.0, from about 7.3 to about 7.7, from about 7.3 to about 7.6, from about 7.3 to about 7.5, from about 7.4 to about 7.7, from about 7.4 to about 7.6, from about 7.4 to about 7.5, or from about 7.5 to about 7.7 or about 7.3, about 7.4, about 7.5, about 7.6, or about 7.7.

The conductivity target of the equilibration (mS/cm) may be from about 6 mS/cm to about 12 mS/cm. The conductivity target of the equilibration (mS/cm) is preferably from about 7.5 mS/cm to about 9.5 mS/cm. The conductivity target may be from about 7.7 mS/cm to about 9.3 mS/cm, from about 7.9 mS/cm to about 9.1 mS/cm, from about 8.1 mS/cm to about 8.9 mS/cm, from about 8.2 mS/cm to about 8.8 mS/cm, from about 8.3 mS/cm to about 8.7 mS/cm, from about 8.4 mS/cm to about 8.6 mS/cm, from about 8.0 mS/cm to about 8.5 mS/cm, from about 8.3 mS/cm to about 8.6 mS/cm, from about 8.3 mS/cm to about 8.5 mS/cm, from about 8.4 mS/cm to about 8.8 mS/cm, or about 8.5 mS/cm.

Loading of the eluate onto the AEX/HIC support is carried out at a temperature, in a volume, and for a time suitable to allow for maximal adsorption of impurities to the N- Benzyl-N-methyl ethanol amine ligand. The monoclonal antibodies preferably do not interact with the ligand, such that the monoclonal antibodies flow through the support during chromatography. Allowing the eluate to flow through the AEX/HIC support results in a flow-through pool comprising the monoclonal antibody. This flow-through pool includes fewer contaminating materials (e.g., residual Protein A, host cell proteins, DNA, etc.) relative to the Protein A eluate loaded onto the support.

It is believed that about 10% to about 20% of the acidic charge variants of the monoclonal antibodies may adsorb to the ligand during the AEX/HIC chromatography step. Thus, for example, if there are about 25% by weight of acidic charge variants in the initial monoclonal antibody preparation (as expressed by the cells), there may remain about 20- 23% of acid charge variants in the flow-through from the AEX/HIC column.

Following the intermediate purification step (e.g., AEX/HIC chromatography), the monoclonal antibody may be still further purified with a third chromatography step.

Preferably, the chromatography employed in this step comprises cation exchange chromatography (CEX). The level of impurities in the AEX/HIC flow through may be determined, and the CEX conditions may be adjusted to attain the desired level of clearance of the remaining impurities. A goal in this regard is to match the level of impurities in the final biosimilar product to the level of impurities in the reference product. For example, the wash conditions in the CEX may be adjusted to clear a higher or lower percentage of charge variants from the AEX/HIC flow through.

The AEX/HIC flow through may be loaded onto a CEX support. In some preferred aspects, the CEX support comprises a sulfapropyl ligand. A non-limiting example of a suitable media includes CAPTO ^® SP ImpRes media. In some aspects, the chromatography media comprises a support comprising a carboxymethyl, phosphate, sulfoethyl, or sulfonate ligand. The ligand may be linked to any suitable support, which may comprise an agarose, ceramic, hydrophilic polymer, polymeric bead, polystyrene-divinyl benzene, or polyvinyl ether support. In highly preferred aspects, CEX chromatography is used as the polishing purification step, and follows AEX/HIC chromatography, which is used as the intermediate purification step. In some aspects, CEX/HIC chromatography may be used as the polishing purification step. Using CEX or CEX/HIC chromatography as the polishing purification step (following AEX/HIC chromatography) represents an inversion of the customary practice in the art, which is to use CEX chromatography as the intermediate purification step and AEX/HIC chromatography as the polishing chromatography step. It is believed that this inversion provides for enhanced removal of acid charge variants of the monoclonal antibody relative to carrying out the chromatography steps in the customary order, with CEX chromatography before the AEX/HIC chromatography.

The flow-through pool from the AEX/HIC chromatography step is preferably loaded onto the chromatography support comprising a sulfonate ligand (or carboxymethyl, phosphate, sulfoethyl, or sulfonate ligand) and allowed to flow through the support, whereby the antibodies interact with the sulfonate ligand. The support preferably comprises particles that may be packed into a chromatography column. The support preferably has an antibody binding capacity of from about 20 g/L to about 80 g/L, from about 25 g/L to about 75 g/L, from about 30 g/L to about 70 g/L, from about 30 g/L to about 65 g/L, from about 30 g/L to about 60 g/L, from about 30 g/L to about 55 g/L, from about 30 g/L to about 50 g/L, from about 25 g/L to about 65 g/L, from about 20 g/L to about 60 g/L, from about 25 g/L to about 50 g/L, from about 20 g/L to about 50 g/L, or from about 20 g/L to about 60 g/L.

Prior to loading the flow-through pool onto the CEX support, the support is preferably equilibrated. The equilibration medium may comprise a buffer. The buffer may comprise from about 1 mM to about 100 mM of sodium phosphate. The buffer may comprise from about 5 mM to about 50 mM of sodium phosphate. The buffer may comprise from about 10 mM to about 40 mM of sodium phosphate, from about 15 mM to about 35 mM of sodium phosphate, from about 15 mM to about 30 mM of sodium phosphate, from about 17 mM to about 28 mM of sodium phosphate, from about 10 mM to about 30 mM of sodium phosphate, from about 20 mM to about 30 mM of sodium phosphate, from about 20 mM to about 28 mM of sodium phosphate, from about 20 mM to about 25 mM of sodium phosphate, from about 15 mM to about 25 mM of sodium phosphate, or from about 24 mM to about 25 mM of sodium phosphate, or about 25 mM of sodium phosphate. As an alternative to sodium phosphate, the buffer may comprise a citrate salt, a succinate salt, MES, Bis-Tris, HEPES, or histidine. The pH of the buffer may be from about 5.0 to about 7.0, from about 5.2 to about 6.8, from about 5.6 to about 6.8, from about 5.6 to about 6.6, from about 5.8 to about 6.6, from about 5.9 to about 6.5, from about 6.0 to about 6.4, from about 6.1 to about 6.3, or about 6.2. The pH of the buffer may be about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

The conductivity target of the equilibration (mS/cm) is preferably from about 1 mS/cm to about 4 mS/cm. The conductivity target may be from about 1 mS/cm to about 3.6 mS/cm, from about 1 mS/cm to about 3 mS/cm, from about 1.5 mS/cm to about 3.5 mS/cm, from about 2 mS/cm to about 3 mS/cm, or from about 2 mS/cm to about 2.5 mS/cm, or about 2.3 mS/cm.

Loading of the flow-through pool comprising the monoclonal antibody onto the CEX support is carried out at a temperature, in a volume, and for a time suitable to allow for maximal adsorption of the monoclonal antibodies to the ligand support. Undesired materials that do not adsorb to the ligand support flow through the support during chromatography. To further remove undesired materials that adhere to the ligand, the antibody- adsorbed support is preferably washed. The washing removes a significant percentage of acid charge variants in the flow-through pool. Any suitable number of washes may be used, and the wash may comprise a buffer and sufficient stringency to remove acid charge variants, but not a substantial portion of non-variant antibodies from the ligand. It is preferable that the chromatography support be washed at least three times for acidic species removal.

The first wash for acidic species removal preferably comprises the same buffer used to equilibrate the CEX support. Thus, for example, the antibody-adsorbed CEX support may be washed with a buffer comprising a pH and a conductivity target as in the column equilibration.

The second wash for acidic species removal may optionally additionally comprise a salt, such as sodium chloride. Thus, the second wash solution may comprise sodium phosphate and sodium chloride. The wash may comprise sodium phosphate at any concentration (mM) described or exemplified herein. As an alternative to sodium phosphate, the wash may comprise a citrate salt, a succinate salt, MES, Bis-Tris, HEPES, or histidine. If sodium chloride is included, the wash may comprise from about 1 mM to about 100 mM of sodium chloride, from about 30 mM to about 60 mM of sodium chloride, from about 35 mM to about 55 mM sodium chloride, from about 40 mM to about 50 mM sodium chloride, from about 41 mM to about 49 mM sodium chloride, from about 42 mM to about 48 mM sodium chloride, from about 43 mM to about 47 mM sodium chloride, from about

43 mM to about 46 mM sodium chloride, from about 43 mM to about 45 mM sodium chloride, from about 42 mM to about 45 mM sodium chloride, from about 42 mM to about

44 mM sodium chloride, or from about 43 mM to about 44 mM sodium chloride. The wash may comprise about 40 mM sodium chloride, about 41 mM sodium chloride, about 42 mM sodium chloride, about 43 mM sodium chloride, about 44 mM sodium chloride, about 45 mM sodium chloride, about 46 mM sodium chloride, about 47 mM sodium chloride, or about 48 mM sodium chloride. About 44 mM sodium chloride is preferred. The pH of the wash may be from about 5.0 to about 7.0, from about 5.2 to about 6.8, from about 5.6 to about 6.8, from about 5.6 to about 6.6, from about 5.8 to about 6.6, from about 5.9 to about 6.5, from about 6.0 to about 6.4, from about 6.1 to about 6.3, or about 6.2. The pH may be about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

The conductivity target of the second wash (mS/cm) is preferably from about 6.6 mS/cm to about 7.6 mS/cm. The conductivity target may be from about 3 mS/cm to about 5.6 mS/cm, from about 3.6 mS/cm to about 5.6 mS/cm, from about 4 mS/cm to about 6 mS/cm, from about 4 mS/cm to about 5.6 mS/cm, from about 4.2 mS/cm to about 5.8 mS/cm, from about 4.3 mS/cm to about 5.5 mS/cm, from about 4.4 mS/cm to about 4.6 mS/cm, from about 4.5 mS/cm to about 5.5 mS/cm, from about 5 to about 7, from about 6 to about 7, from about 6.7 mS/cm to about 7.5 mS/cm, from about 6.8 mS/cm to about 7.4 mS/cm, from about 6.9 mS/cm to about 7.3 mS/cm, or from about 7 mS/cm to about 7.2 mS/cm, about 7.1 mS/cm or about 7.2 mS/cm.

The third wash for acidic species removal preferably comprises the same buffer used to equilibrate the CEX support. Thus, for example, the antibody-adsorbed CEX support may be washed a third time with a buffer comprising a pH and a conductivity target as in the column equilibration. The third wash preferably does not include sodium chloride. The third wash may effectively quench the acidic species removal from the second wash, as the conductivity target is lowered.

Washing during the CEX polishing chromatography step preferably removes acid charge variants of the monoclonal antibodies. A balance is preferred, in which a desired amount of acid charge variants is removed from the antibody population, yet a maximal amount of desired (non-variant) monoclonal antibodies are retained on the support for subsequent elution. It was observed that a more stringent wash could remove additional acid charge variants, but such a more stringent wash also removed more desired (non- variant) monoclonal antibodies, and conversely, a less stringent wash could retain more of the desired population of non-variant antibodies, but would remove fewer acid charge variants. It was further observed that monitoring the UV absorbance (e.g., at UV A280) in the CEX chromatography system during the second wash can facilitate achievement of an appropriate balance between antibody (non-variant) yield and acid charge variant removal. Column volume measurements may be used in the alternative to UV absorbance. When this balance is achieved, the stringency in the second wash is quenched by the initiation of the third wash.

Thus, for acid charge variant removal, the polishing chromatography step preferably includes monitoring of UV absorbance, and determining the peak of absorbance during the second wash, then determining when the absorbance diminishes from about 5% to about 20%, and then quenching the salt (sodium chloride) stringency by washing the CEX support with the third wash (e.g., equilibration) buffer. Preferably, the third wash is initiated when the absorbance diminishes from about 5% to about 15%, or from about 5% to about 14%, or from about 5% to about 13%, or from about 5% to about 12%, or from about 5% to about 11%, or from about 5% to about 10%, or from about 6% to about 15%, or from about 6% to about 14%, or from about 6% to about 13%, or from about 6% to about 12%, or from about 6% to about 11%, or from about 6% to about 10%, or from about 7% to about 15%, or from about 7% to about 14%, or from about 7% to about 13%, or from about 7% to about 12%, or from about 8% to about 15%, or from about 8% to about 14%, or from about 8% to about 13%, or from about 9% to about 15%, or from about 9% to about 14%, or from about 9% to about 13%, or from about 10% to about 15%, or from about 10% to about 14% of the peak absorbance units.

The CEX polishing step may also remove basic charge variants. Steps for removal of basic charge variants may be in addition to the steps for removal of acid charge variants, or in the alternative to steps for removal of acid charge variants (e.g., CEX polishing may be employed to remove acidic species, basic species, or both acidic and basic species). I n some preferred aspects, both acid and basic charge variants are removed by CEX chromatography polishing. Basic charge variants may be removed by a gradient or stepwise elution, and not via washing of the support.

Prior to gradient elution for basic species removal, the support may be washed using the same buffer used to equilibrate the CEX support. Thus, for example, the antibody- adsorbed CEX support may be washed with a buffer comprising from about 5 mM to about 50 mM of sodium phosphate at a pH of from about 6.4 to about 7.2, and a conductivity target of from about 1 mS/cm to about 4 mS/cm, as set forth above. The wash may comprise from about 1 mM to about 100 mM of sodium phosphate. The buffer may comprise from about 5 mM to about 50 mM of sodium phosphate. The buffer may comprise from about 10 mM to about 40 mM of sodium phosphate, from about 15 mM to about 35 mM of sodium phosphate, from about 15 mM to about 30 mM of sodium phosphate, from about 17 mM to about 28 mM of sodium phosphate, from about 10 mM to about 30 mM of sodium phosphate, from about 20 mM to about 30 mM of sodium phosphate, from about 20 mM to about 28 mM of sodium phosphate, from about 20 mM to about 25 mM of sodium phosphate, from about 15 mM to about 25 mM of sodium phosphate, or from about 24 mM to about 25 mM of sodium phosphate, or about 25 mM of sodium phosphate. As an alternative to sodium phosphate, the buffer may comprise a citrate salt, a succinate salt, MES, Bis-Tris, HEPES, or histidine. The pH of the buffer may be from about 6.4 to about 7.2, from about 6.4 to about 7.1, from about 6.5 to about 7.1, from about 6.5 to about 7.0, from about 6.5 to about 6.9, from about 6.6 to about 7.0, from about 6.6 to about 6.9, from about 6.7 to about 6.9, from about 6.8 to about 6.9, from about 6.6 to about 6.8, from about 6.5 to about 6.7, or from about 6.8 to about 7.0. The pH of the buffer may be about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, or about 7.2.

The conductivity target of the wash (mS/cm) is preferably from about 1 mS/cm to about 4 mS/cm. The conductivity target may be from about 1 mS/cm to about 3.6 mS/cm, from about 1 mS/cm to about 3 mS/cm, from about 1.5 mS/cm to about 3.5 mS/cm, from about 2 mS/cm to about 3 mS/cm, or from about 2 mS/cm to about 2.5 mS/cm, or about 2.3 mS/cm.

Following washing (e.g., under an acidic species and/or basic species removal protocol), the monoclonal antibodies are eluted from the CEX support. Elution may be carried out at a temperature, in a volume, and for a time suitable to allow for maximal elution yield of the monoclonal antibodies from the ligand support.

The elution buffer may comprise sodium phosphate at any concentration (mM) described or exemplified herein. As an alternative to sodium phosphate, the elution buffer may comprise a citrate salt, a succinate salt, MES, Bis-Tris, HEPES, or histidine. The elution buffer also comprises from about 75 mM to about 500 mM sodium chloride, from about 100 mM to about 500 mM, from about 80 mM to about 120 mM of sodium chloride, from about 80 mM to about 110 mM sodium chloride, from about 80 mM to about 100 mM sodium chloride, from about 90 mM to about 120 mM sodium chloride, from about 90 mM to about 110 mM sodium chloride, from about 90 mM to about 105 mM sodium chloride, from about 95 mM to about 120 mM sodium chloride, from about 95 mM to about 115 mM sodium chloride, from about 95 mM to about 110 mM sodium chloride, from about 95 mM to about 105 mM sodium chloride, or from about 98 mM to about 102 mM sodium chloride. The elution buffer may comprise about 80 mM sodium chloride, about 85 mM sodium chloride, about 90 mM sodium chloride, about 95 mM sodium chloride, about 96 mM sodium chloride, about 97 mM sodium chloride, about 98 mM sodium chloride, about 99 mM sodium chloride, about 100 mM sodium chloride, about 101 mM sodium chloride, about 102 mM sodium chloride, about 103 mM sodium chloride, about 104 mM sodium chloride, or about 105 mM sodium chloride. About 100 mM sodium chloride is preferred. As an alternative to sodium chloride, ammonium sulfate or sodium sulfate may be used.

The pH of the elution buffer may be from about 5.0 to about 7.0, from about 5.8 to about 6.6, from about 5.9 to about 6.5, from about 6.0 to about 6.4, from about 6.1 to about 6.3, or about 6.2. The pH of the buffer may be about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6. The conductivity target of the elution (mS/cm) is preferably from about 10 mS/cm to about 14 mS/cm. The conductivity target may be from about 11 mS/cm to about 14 mS/cm, from about 11 mS/cm to about 13 mS/cm, from about 12 mS/cm to about 14 mS/cm, from about 12 mS/cm to about 13 mS/cm, from about 12.3 mS/cm to about 13.3 mS/cm, from about 12.5 mS/cm to about 13.1 mS/cm, or from about 12.6 mS/cm to about 13 mS/cm, or about 12.8 mS/cm. Elution buffer where only basic charge variants of the antibody are removed may be run using a linear gradient or employing a step elution in an isocratic mode.

It is preferred that acidic species are removed via washing with a higher stringency as described above. While the column may be washed during a basic species removal, the washing is less stringent than for acidic species removal; instead, basic species are removed via increasing stringency in the elution buffer. If acidic species have been washed from the support and removal of basic species is not desired, the antibody is eluted from the CEX column with the elution buffer at a fixed concentration of sodium chloride. If removal of basic species is desired (whether or not acidic species have been washed from the support), the antibody is eluted from the CEX column using elution buffer with an increasing concentration of sodium chloride. The increase in sodium chloride may be achieved via a linear gradient, whereby the elution buffer at the inception of the elution stage has a lower concentration of sodium chloride, and as the elution stage progresses, sodium chloride is added to the elution buffer at a substantially constant rate in order to attain the highest desired concentration of sodium chloride for antibody elution. Alternatively, the increase in sodium chloride may be achieved in steps, whereby the elution buffer at the inception of the elution stage has a lower concentration of sodium chloride, and after a period of time elapses, the elution buffer is changed to include a buffer with a higher concentration of sodium chloride. Any suitable number of stepwise enhancements of the sodium chloride concentration may be used, with two or three step increases being preferred.

It is believed that about 25% to about 60% of the remaining acidic charge variants of the monoclonal antibodies are removed by the second wash during the CEX

chromatography step. Thus, for example, if there are about 20-23% by weight of acid charge variants in the flow-through from the AEX/HIC column, there will remain about 8% to about 20% by weight of acid charge variants in the eluate from the CEX column. Preferably, about 10% to about 14% by weight of acid charge variants are in the eluate from the CEX column, as measured by CEX-HPLC. Preferably, about 15% to about 17% by weight of acid charge variants are in the eluate from the CEX column, as measured by icIEF. In some aspects, about 10% to about 12% by weight of acid charge variants are in the eluate from the CEX column, as measured by either CEX-HPLC or icIEF. In some aspects, about 11% to about 13% by weight of acid charge variants are in the eluate from the CEX column, as measured by either CEX-HPLC or icIEF. In some aspects, about 12% to about 14% by weight of acid charge variants are in the eluate from the CEX column, as measured by either CEX- HPLC or icIEF. In some aspects, about 14% to about 18% by weight of acid charge variants are in the eluate from the CEX column, as measured by either CEX-HPLC or icIEF. In some aspects, about 13% to about 19% by weight of acid charge variants are in the eluate from the CEX column, as measured by either CEX-HPLC or icIEF. In some aspects, about 14% to about 17% by weight of acid charge variants are in the eluate from the CEX column, as measured by either CEX-HPLC or icIEF. In some aspects, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, or about 17% by weight of acid charge variants are in the eluate from the CEX column, as measured by either CEX-HPLC or icIEF. It is believed that about 25% to about 60% of the remaining basic charge variants of the monoclonal antibodies are removed by using a linear gradient or step elution process to remove the tailing shoulder that is evident during the elution phase of the CEX

chromatography step. Thus, for example, there will remain about 5% to about 35% by weight of basic charge variants in the eluate from the CEX column. Preferably, about 10% to about 30% by weight of basic charge variants are in the eluate from the CEX column, as measured by any suitable technique, for example, CEX-HPLC or icIEF. In some aspects, about 15% to about 25% by weight of basic charge variants are in the eluate from the CEX column, as measured by any suitable technique. In some aspects, about 15% to about 30% by weight of basic charge variants are in the eluate from the CEX column, as measured by any suitable technique. In some aspects, about 12% to about 27% by weight of basic charge variants are in the eluate from the CEX column, as measured by any suitable technique. In some aspects, about 18% to about 23% by weight of basic charge variants are in the eluate from the CEX column, as measured by any suitable technique. In some aspects, about 20% to about 25% by weight of basic charge variants are in the eluate from the CEX column, as measured by any suitable technique. In some aspects, about 19% to about 22% by weight of basic charge variants are in the eluate from the CEX column, as measured by any suitable technique. In some aspects, about 17% to about 21% by weight of basic charge variants are in the eluate from the CEX column, as measured by any suitable technique. In some aspects, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% by weight of basic charge variants are in the eluate from the CEX column, as measured by any suitable technique.

The CEX-HPLC testing method monitors charge heterogeneity, including C-terminal variants, via weak cation exchange chromatography at a pH range of 5.6 to 8.0. The method may employ, for example, a Dionex ProPac™ WCX-10 column with a MES/sodium

phosphate/sodium chloride gradient, with UV absorbance monitored at 280 nm. Distinct peaks eluting after the main peak are considered basic species, and peaks eluting prior to the main peak are considered acidic species.

Imaged capillary isoelectric focusing (icIEF) is a high-resolution imaging technique for protein separation and detection based on differences in their isoelectric points (pi). The icIEF method uses a Protein Simple iCE3 system, together with Alcott autosampler.

Following addition of the protein sample to a mixture of ampholytes, stabilizers

methylcellulose and pi markers, the solution is injected into a fluorocarbon (FC)-coated capillary. The protein sample is then separated and analyzed using two steps of focusing. The system first pre-focusing step mobilizes the samples in the mixture of ampholytes at 1,500 kV. Next, the system second focusing applies a higher voltage at 3,000 kV across the capillary forming a pH gradient through the introduction of hydronium ions from the anolyte and hydroxyl ions from the catholyte at the opposite end of the capillary. The pH gradient forms at the capillary ends and then progresses toward the center of the capillary where both anodic and cathodic sides merge. Bidirectional focusing of the protein sample occurs by allowing the test article proteins to migrate and finally settle at their specific pi values. As a result, the pH gradient is titrated from basic to acidic and the protein charge variants can be separated according to the differences in pi values and are then detected at 280 nm as the proteins obtain a positive charge and migrate toward the cathode by whole column imaging using a CCD camera.

The eluate from the CEX column contains purified monoclonal antibodies with a minimal amount of acid charge variants and/or a minimal amount of basic charge variants. The eluate may be further processed in order that the monoclonal antibodies may be in a form suitable for therapeutic administration, for example, to a human patient. The further processing may include any combination of nanofiltration, concentration, and diafiltration of the eluate.

Nanofiltration may comprise filtering the eluate from the CEX column (including the monoclonal antibodies and remaining charge variants) through a filter having an about 15 nm pore size, or about 20 nm pore size. Nanofiltration may remove viruses and precipitates present in the eluate.

Concentration may comprise ultrafiltration, using a membrane with an appropriate molecular weight cutoff. Both ultrafiltration and diafiltration may be carried out according to standard methodologies, using standard equipment as is practiced in the art.

The monoclonal antibodies, pooled and purified according to the chromatography, virus inactivation, filtration, and concentration modalities described above may be formulated for storage and therapeutic administration to patients. The monoclonal antibody formulation may be for subcutaneous administration to patients. In some aspects, the monoclonal antibody pool comprises about 51% to about 75% by weight of non-variant antibodies (not basic species or acid species), from about 10% to about 14% by weight of acid charge variants of the monoclonal antibody, and from about 15% to about 35% by weight of basic charge variants of the monoclonal antibody. This antibody pool may be formulated with an aqueous buffer and a non-ionic surfactant, and the formulation may comprise from about 30 mg to about 55 mg of the monoclonal antibody pool (to include the non-variant antibodies and the charge variants). In some aspects, the formulation comprises from about 35 mg to about 45 mg of the antibody pool. In some aspects, the formulation comprises from about 45 mg to about 55 mg of the antibody pool. In some preferred aspects, the formulation comprises from about 40 mg to about 50 mg of the antibody pool. In some preferred aspects, the formulation comprises about 40 mg of the antibody pool. In some preferred aspects, the formulation comprises about 45 mg of the antibody pool. In some highly preferred aspects, the formulation comprises about 50 mg of the antibody pool. The antibody is preferably dosed at a total of 40 mg and about 0.8 mg/mL of the antibody.

The buffer may comprise from about 0.8 mM to about 1.2 mM of sodium acetate trihydrate, or from about 0.9 mM to about 1.1 mM of sodium acetate trihydrate, or about 1 mM of sodium acetate trihydrate. The buffer may comprise from about 201 mM to about 205 mM of mannitol, or from about 202 mM to about 204 mM of mannitol, or about 203 mM of mannitol. The buffer may comprise from about 17 mM to about 21 mM of glacial acetic acid, or from about 18 mM to about 20 mM of glacial acetic acid, or about 19 mM of glacial acetic acid. The buffer may comprise from about 25 mM to about 27 mM of sodium chloride, or about 26 mM of sodium chloride, or about 27 mM of sodium chloride, or about 26.35 mM of sodium chloride.

The buffered antibody formulation may include polysorbate 80 as a non-ionic surfactant. In some aspects, the formulation comprises from about 0.08% (v/v) to about 0.12% (v/v) of polysorbate 80. In some aspects, the formulation comprises from about 0.09% (v/v) to about 0.11% (v/v) of polysorbate 80. In some aspects, the formulation comprises about 0.1% (v/v) of polysorbate 80. In a detailed aspect, a buffered antibody formulation comprises (a) about 40 mg to about 60 mg, preferably about 50 mg of the monoclonal antibody pool (to include the non- variant antibodies and the charge variants), (b) a buffer comprising about 1 mM of an acetate salt, preferably sodium acetate trihydrate, about 203 mM of mannitol, about 19 mM of glacial acetic acid, and about 26.35 mM of sodium chloride, and (c) about 0.1% (by volume) of polysorbate 80. The buffered monoclonal antibody formulation has a pH of from about 5.1 to about 5.3, preferably about 5.2. In some aspects, the formulation comprises from about 35 mg to about 45 mg of the antibody pool. In some aspects, the formulation comprises from about 45 mg to about 55 mg of the antibody pool. In some preferred aspects, the formulation comprises from about 40 mg to about 50 mg of the antibody pool. In some preferred aspects, the formulation comprises about 40 mg of the antibody pool. In some preferred aspects, the formulation comprises about 45 mg of the antibody pool. In some highly preferred aspects, the formulation comprises about 50 mg of the antibody pool. The antibody is preferably dosed at a total of 40 mg and about 0.8 mg/mL of the antibody.

The following examples are provided to describe the invention in greater detail. They are intended to illustrate, not to limit, the invention.

Example 1

Affinity Capture

Protein-A affinity capture step is an inherently robust processing step, with a rich platform performance history. Process impurities such as host cell proteins (HCP) and DNA are removed during the flow-through and wash phases of the step. A low pH buffer eluted the bound adalimumab antibody from the resin and set up the low pH viral inactivation step described in Example 2. Four resins were screened for use using a base process (Tables 1 and 2).

Table 1. Protein A Resins Screened

Bead Size Flow Rate Theoretical

Resin Supplier Matrix

(urn) (cm/h) DBC (g/L)

ABSELECT Rigid

GE 85 100-500 30-35 SURE ^® Agarose

UNOsphere™ Crosslinked 30 (150

Bio-Rad 57 100-600 SUPrA Polymer cm/h)

PROSEP ^®

Millipore Glass 60 500 50 Ultra Plus

Table 2. Protein A Screening Process

Chromatography Step Parameter

Equilibration 20mM sodium phosphate, 150mM sodium chloride, pH 7.2

Sample Loading adalimumab (Residence time = 2 min)

Wash 20mM sodium phosphate, 150mM sodium chloride, pH 7.2

Elution 80mM acetic acid, pH 3.5

CIP 50mM sodium hydroxide, 1M sodium chloride

Re-equilibration 20mM sodium phosphate, 150mM sodium chloride, pH 7.2

During Protein A resin screening, assessment of purity measured by size exclusion chromatography (SEC) as well as product yield were taken into account. Additionally, flow characteristics of the resins (e.g., compressibility, bead matrix and maximum and recommended flow rates) were assessed as they impact resolution of the product and processing times. A summary of the resulting data from the resin screen is shown in Table 3. MabSelect SuRe™ (GE Healthcare) was selected based on an initial dynamic binding capacity (DBC) of 35 g/L as well as a purity of nearly 97% monomer. The product recovery following elution of the material was 99% monomer. The material can be used at flow rates up to 500 cm/h allowing favorable processing times and minimal bead compression.

Table 3. Summary of Protein A Resin Screen

A protein uptake experiment was performed to enable a prediction of dynamic binding capacity at 10% breakthrough while varying residence times (τ) using a calculation of static binding capacity. The uptake study also enables the calculation of the adsorption isotherm for adalimumab with the selected chromatography resin. The design of the experiment is shown in Figure 2. The base process was used previously described in Table 2. The protein uptake study was designed to utilize 96 well format to maximize product and buffer solutions.

Static binding (Q) capacity of adalimumab with MabSelect SuRe ^® was determined using the following Equation 1 and the data is shown in Figure 3. The static binding capacity was then used to generate an adsorption isotherm (Equation 2) which predicts a dynamic binding capacity (qmax) shown in Figure 4

Equation 1. Static binding capacity (Q)

€ , ■ x¥ , .

Q =

Y

Ceiution = adalimumab concentration in eluate pool (mg/mL)

eiution = elution volume (mL)

medium = volume of resin in well (mL)

Equation 2. Adsorption isotherm

¾ - ½ -€ }—

C ₀ = adalimumab concentration (mg/mL)

C = adalimumab concentration in the liquid phase after incubation (mg/mL)

Viiquid = elution volume (mL)

medium = volume of resin in well (mL)

The prediction of dynamic binding capacity from the derivation of the static binding capacity is in agreement with the data generated during the resin screen of 35 g/L binding capacity of adalimumab when using MabSelect SuRe ^® under the chromatographic conditions described. An effort to improve binding capacity was made focusing on the concentration of NaCI used in the equilibration or binding buffer used. Sodium chloride concentrations ranging from 0 to 150 mM were evaluated in the same 20 mM sodium phosphate buffer, pH 7.2 and a residence time of 4 minutes for their impact on binding capacity. The data are shown in Table 4.

Table 4. Impact of NaCI concentration on DBC for adalimumab - MabSelect SuRe ^®

50 4 300 50 59

100 4 300 46.7 54.9

150 4 300 37.1 43.6

A sodium chloride concentration of 50 mM was selected. Experiments to determine the relationship between DBC and residence time and linear velocity are summarized in Figure 5 and Figure 6.

Statistical design of experiments (DoE) was used to optimize all aspects of the Protein-A capture step. Initially forty-eight different elution conditions were evaluated. The experimental design is summarized in Figure 7 with the resultant data represented in Figure 8.

The results indicate that the elution pH range of 3.0 to 3.5 gives a maximum recovery of greater than 95% when the sodium chloride content of the elution matrix is less than 100 mM concentration. Finally, there were no reportable changes in high molecular weight content of the eluate within the pH and sodium chloride ranges tested in this study indicating a robust elution condition. Table 5 outlines the optimized adalimumab capture step. Table 6 summarizes the impurity clearances and product quality achieved using the capture step described above.

Table 5. MabSelect SuRe ^® Capture Step

Table 6. MabSelect SuRe ^® Eluate Impurity Profile

Example 2

Virus Inactivation

The potential for the introduction or presence of adventitious viruses exists when mammalian cell lines are employed in the production of monoclonal antibodies.

Inactivation of these potential adventitious viruses is routinely addressed by the use of orthogonal steps during the purification process that are either specifically designed for or provide viral inactivation or removal as part of their general use.

The first of such steps in the process is the use of a low pH for an extended period of time to inactivate viruses. Changes in aggregation or high molecular weight species (HMW) that may form as a result of the low pH are monitored. The formation of HMW species should be avoided by the use of chemical agents and other parameters (e.g. agitation, duration, temperature) that promote the monomeric form of the mAb. The pH of the eluate collected from the affinity column following each cycle is lowered by the addition of 1 M citric acid, bringing the pH to 3.5 ± 0.1, and holding the material at room temperature for 60 to 70 minutes. Following the inactivation hold period, the pH is raised to 7.5±0.2 by the addition of 3 M Tris. In the event of precipitation of some residual host cell impurities such as host cell proteins and genomic DNA, the inactivated material was depth-filtered to remove any precipitation, followed by a sterilizing grade 0.22 μιη filter. Table 7 shows a summary of chemical agents and parameters for ensuring that the monomeric form of the protein ps well as virus inactivation are maximized.

Table 7. Development Parameters Tested for Low pH Virus Inactivation.

Chemical Agent pH Range Temperature Range Agitation

(°C)

Citric Acid

Adalimumab showed no sensitivity specific to either acetic or citric acid relative to a change in percent monomer. During the process of lowering the pH, a significant amount of precipitation occurred and was determined to be a combination of residual host cell proteins as well as residual DNA. Additionally, the pH of the material had no impact on the aggregation state of the material. However, the pH of the material does have an impact on the viral log reduction value (LRV). The higher the pH during the hold period, the lower the LRV for the operation. Therefore, a pH of 3.5 was selected. Due to the relative stability of the monomer at low pH a hold period at ambient temperature (18-25°C) was used to favor a faster inactivation reaction. Agitation is a parameter that can impact the LRV as well.

Investigating a "worst case" included no agitation to simulate poor or no mixing during the hold period. The conditions selected are shown in Table 8 and were tested in duplicate in a viral spiking study performed to assess the LRV. The viral spiking study was performed with time points of 5, 30 and 60 minute low pH hold times. The data from spiking study is shown in Table 9.

Table 8. Low pH Hold Parameters

Table 9. Viral Spiking Study Results for Low pH Virus Inactivation

Example 3 Intermediate Purification

Intermediate purification of the adalimumab antibody was accomplished via mixed mode chromatography using CAPTO ^® Adhere resin as a stationary media. CAPTO ^® Adhere is a strong anion exchanger with multimodal functionality. The ligand, N-Benzyl-N-methyl ethanol amine exhibits ionic interactions. The most significant of these interactions are hydrogen bonding and hydrophobic interaction. The step was run in flow-through mode and had a dynamic binding capacity of 75-125 g adalimumab/L packed resin while clearing significant amounts of process-related impurities such as leached Protein A, host cell proteins, residual genomic host cell DNA; product variants such as high molecular weight species (antibody dimer and higher order aggregates) and acidic charge variants; as well as potential viruses.

The mixed mode resin was conducted in a flow-through mode whereby the adalimumab material will not interact with the stationary resin while process and product related impurities are retained, to varying degrees, by the resin thereby achieving additional purification. Loading conditions were screened initially to determine the optimal mobile phase conditions to facilitate maximum impurities clearance while achieving an acceptable yield. The base screening conditions are outlined in Table 10.

Table 10. Mixed mode loading conditions screening process.

The results of the initial scouting experiment were used to determine the

parameters of subsequent DoE. The following DoE (Table 11) was designed to map the operating space and relationship between operating parameters (e.g,. pH, conductivity mass loading) and responses (e.g., yield, impurity clearances).

The pH at the UV-280nm elution peak apex can be used to define the pH minimum in the subsequent design. The pH maximum in the design can then be estimated to be approximately 2 pH units above the minimum. In this case, the pH at the UV apex is 6.3 thus the next series of experiments were set within a pH range of 6.0 to 8.0 (Table 11A). The design of experiments was executed at 100 g adalimumab/L resin.

Table 11A. Mixed mode chromatography binding condition DoE parameters

The results of the DoE are shown in Table 11B and Figures 9A and 9B. Figure 9A shows the step yield resulting from relationship between pH and conductivity when adalimumab is loaded as previously described. Figure 12B shows the reduction of high molecular weight species as a function of pH and conductivity relative to the starting material. Figure 10 shows a similar contour plots related to both change in acidic charge variants as well as CHO host cell protein clearance. Data from this study also showed that the mixed mode chromatography step is capable of clearing residual host cell DNA to below the level of quantitation of the assay.

Table 11B. Effect of pH and Conductivity on adalimumab using CAPTO ^® Adhere

Chromatography.

8 6 30 82 25.6 0.5 1.65 0.55 <LOQ 25.8

9 6 16 98 25.6 0.5 1.92 0.28 <LOQ 23.2

10 7 30 91 25.1 1 0.55 1.65 <LOQ 30.7

Using the process parameters in Table 12, acidic charge variant and high molecular weight clearance profiles over a range of binding capacities (50 - 200 g adaiimuma /L resin) were generated in Figure 11 and Figure 12. Yields were also trended in both figures. Recoveries increased from 67 - 85% with increasing mass loads. In contrast, clearance of acidic species and high molecular weight decreased with increasing binding capacity.

Table 12. Mixed mode varying binding capacity process conditions

An additional study was conducted to determine the effectiveness of several buffer systems of the performance of chromatography step under optimal pH and conductivity. The data are shown in Table 13. Buffer systems including bis-tris, sodium phosphate and HEPES were tested side by side at common pH and conductivities of 7.5 and 8.5 mS/cm respectively. Recovery as well as aggregate and acidic charge variant clearances was of primary interest in the study. The data indicate that while there are only subtle differences (within the variability of the assays used to test) there is an unexpected and significant improvement in recovery of adalimumab when HEPES is employed as the mobile phase. Table 13. Performance of buffer systems used on CAPTO ^® Adhere purification of adalimumab.

AACV (%) -3 -3.2 -2.1

ΔΗΜ\Λ/ (%) -2.2 -2.3 -2.1

The development and data described above regarding the intermediate chromatography step maps out a design space that shows the impact pH, conductivity and resin mass loading have on process (yield) and product parameters (HMW, HCP, rDNA and acidic charge species). The contour maps shown above are indicative of a process that is robust and will produce adalimumab with increased purity (Table 14) and can be further polished if necessary. Table 14 shows the preferred mobile phase conditions for the intermediate polishing of adalimumab.

Table 14. CAPTO ^® Adhere Flow-Through Impurity Profile

Table 15. CAPTO ^® Adhere Intermediate Chromatography Step

Example 4 Polishing Purification - Acidic Species

The cation exchange chromatography is performed using CAPTO ^® SP ImpRes resin. This step is primarily for the reduction of acidic charge variants, leached Protein A, host cell proteins and potential viruses. The step is run in bind and elute mode and has a dynamic binding capacity of 25-50 g adalimumab/L packed resin (up to 60 g of binding has been achieved). Cation exchange chromatography is used primarily to reduce the remaining acidic charge species to within range of the reference product (HUMIRA ^® adalimumab, Abbvie Biotechnology Ltd. Corp.). Additional clearance of process and product related impurities (HMW, HCP, rPA, rDNA) as well as potential viruses can also be achieved during this step.

A series of experiments were performed to determine the most effective processing pH to bind and elute adalimumab. Six runs were conducted using pH values ranging from 7.5 - 5.0 in 0.5 unit increments with process conditions outlined in Table 16.

Table 16. Initial Polishing Chromatography pH Conditions Screening Process

As the pH of the processing conditions decrease, adalimumab drifts further from its pi into an increasingly acidic environment. Thus, the protein requires a greater salt content to elute, which causes the resulting shift in eluates as pH decreases. A pH value of 6.2 was determined to be the processing condition best suited to achieve efficient binding and elution of adalimumab.

A follow up experiment was performed using higher phosphate content in the mobile phase to attempt to minimize the pH drift during the salt addition when creating the conductivity gradient necessary for elution of adalimumab. The first run repeated the standard lOmM sodium phosphate used in the pH screening study, while the second run implemented a stronger phosphate buffer of 25mM. Processing conditions are in Table 17.

Table 17. Polishing Chromatography Mobile Phase Strength Processing Conditions

During the conductivity gradient elution, less than a 0.5 pH unit difference was observed when using the stronger phosphate buffer of 25mM. The greater buffering capacity of the 25mM sodium phosphate mobile phase was attributed to reducing the pH fluctuation during elution and used moving forward.

The following experiment was designed to evaluate how varying conductivities affect separation of the charge variant species in adalimumab. Protein will be bound and eluted in a stepwise fashion by pumping 1M sodium chloride at increasing increments to achieve salt environments of roughly 20, 40, 60, 80, and lOOmM sodium chloride. Each increment will last for 5 CVs. A final step of 350mM sodium chloride will be targeted for 10 CVs to elute any remaining protein from the resin. Process parameters are outlined in Table 18.

Table 18. Polishing Chromatography Conductivity Step Elution Conditions

Protein begins to elute during the 40mM sodium chloride step and reaches a peak maximum. Upon its descent, the salt step increased to 60mM sodium chloride, where we see another peak elute from the column. Two more additional peaks, although slightly smaller in area, elute during the 80 and lOOmM sodium chloride steps. When the final step of 350mM sodium chloride was executed, the remainder of the protein eluted.

Samples were collected at each step and tested for charge variant species and polydispersity, with results in Table 19. Fractions A3 and A4, corresponding to salt contents of 40 and 60mM sodium chloride, contained the highest percentage of acidic species (59.2 and 22.8%, respectively). As the salt content increased further, the acidic species consistently decreased to just 0.3% in the final fraction of 350mM sodium chloride. SEC results showed an opposing trend, with aggregation formation occurring with increasing salt concentrations. The results were successful in demonstrating that the acidic species can be separated by slight increases in conductivity and allow the remaining protein, which is now significantly less rich in acidic character, to be eluted in a higher salt environment.

Table 19. Polishing Chromatography Conductivity Step Elution Impurity Profile

A number of runs were performed to determine the most effective wash parameters to achieve sufficient acidic species removal while minimizing yield loss over the polishing chromatography step. Salt contents of 44 and 60mM sodium chloride were tested based on the results of the conductivity step gradient elution study, and the duration of the wash was varied. The yield and acidic charge variant clearance (AACV) were calculated and tested for each run. Run conditions are shown in Table 20.

Table 20. Polishing Chromatography Varying Salt and Duration of Wash Step Chromatography Step Parameter

Equilibration 25mM sodium phosphate, pH 6.2

Sample Loading adalimumab (Residence time = 7-8 min, binding capacity = 30 g/L resin)

Wash 1 25mM sodium phosphate, pH 6.2

Wash 2 25mM sodium phosphate 44/60mM sodium chloride, pH 6.2

Wash 3 25mM sodium phosphate, pH 6.2

Elution 25mM sodium phosphate 100/350mM

sodium chloride, pH 6.2

Strip 1M sodium chloride

Re-equilibration 25mM sodium phosphate, pH 6.2

CIP IN sodium hydroxide

Re-equilibration 25mM sodium phosphate, pH 6.2

Results of the varying salt and wash duration for acidic variant removal are shown in Table 21. High yields were attained using 60mM sodium chloride for a 1.5 CV duration, but minimal acidic charge variant clearance was achieved. Greater clearance of acidic species was achieved by increasing the wash duration to 2 CVs (5.6 - 8.0), but the broad range of clearance with just a 0.5 CV increment in wash duration was not desirable to produce a consistent process. Decreasing the salt content to 44mM sodium chloride allowed the wash to run for a broader duration (3 - 5 CVs) to achieve a tighter range of acidic clearance comparable to the 60mM sodium chloride. Employing a wash duration of 3 - 5 CVs in 44mM sodium chloride condition consistently cleared 5 - 7% of the acidic charge variant with yields ranging from approximately 60 - 70%.

Table 21. Polishing Mode Chromatography Varying Salt and Duration of Wash Step Results

To create a more robust wash step, the duration of the wash was altered from a volume to UV based criteria. Rather than running the wash using a CV collection, it is more appropriate to standardize to a UV A280 value for the duration of the wash. This will help reduce variation between runs with regards to the amount of protein loaded. Process conditions are shown in Table 22.

Table 22. Polishing Chromatography Varying Salt and Duration of Wash Step

For the 44mM sodium chloride wash 2 utilizing a 4 CV wash duration, adalimumab rich in acidic species began to elute, reached a UV A280 peak maximum of 1297 mAU, and decreased to 1151 mAU upon completing 4 CVs. The UV at which wash 2 concluded was calculated to be approximately 90% of the peak maximum. This value was used moving forward to standardize the end of wash 2 using 44mM sodium chloride to reduce the acidic charge variant.

The current elution buffer, 25mM sodium phosphate 350mM sodium chloride, showed an increase in high molecular weight of the eluate over the polishing step. The high salt environment may be the contributing factor to increased aggregation and is also known to cause pressure issues during the viral filtration step, which occurs after the polishing chromatography in the adalimumab process. A series of experiments were conducted using 350, 150, and lOOmM sodium chloride in the elution buffer with yield and SEC being monitored. Process conditions are in Table 23.

Table 23. Polishing Chromatography Varying Salt Elution Buffer Conditions

Wash 2 25mM sodium phosphate 60mM sodium chloride, pH 6.2

Wash 3 25mM sodium phosphate, pH 6.2

Elution 25mM sodium phosphate 350/150/100mM sodium chloride, pH 6.2

Strip 1M sodium chloride

Re-equilibration 25mM sodium phosphate, pH 6.2

CIP IN sodium hydroxide

Re-equilibration 25mM sodium phosphate, pH 6.2

Results from varying salt content in the elution buffer are in Table 24. As the molarity of salt decreases, aggregate was reduced. At the lowest salt condition tested, no high molecular weight was created over the polishing step.

Table 24. Polishing Chromatography Varying Salt Content of Elution Buffer Results

Wash 2 was designed to remove a significant portion of the acidic species inherent to adalimumab. Because of this, yields over this step range from just 60 - 70%. A DoE was designed over a range of pH and conductivities in attempt to minimize protein loss while retaining the high reduction of acidic variant. The ranges were attained by conducting two gradient elution runs, the first of which eluted adalimumab via a pH gradient, the second using conductivity. Using offline pH and conductivity meters to verify, the UV A280 peak max of the eluate occurred at a pH of 7.5 and conductivity of 13 mS/cm, respectively. From these results, the corresponding pH and conductivity ranges for the DoE are outlined in Table 25. Table 25. Polishing mode chromatography wash 2 condition DoE parameters and Data

The DoE described above was designed to elucidate a set of robust parameters that a cation exchange chromatography step can be operated to provide maximum yield while reducing product and process related impurities. The product quality attributes monitored most closely during this step are the charge heterogeneity profile and high molecular weight species. Modulation of the pH and conductivity conditions in the second washing step is important for reducing the amount of acidic charge species in the monoclonal antibody material.

As with most chromatography conditions, a balance must be found between the required amount of impurity reduction and material recovery (yield) for the operation. This step has been designed to separate species of adalimumab with only very subtle changes in charge (acidic species) from the main species population. Table 25 shows the yield, high molecular weight, and acidic charge clearance data that have been generated. It is evident that even small variations in pH (5.0-6.8) and conductivity (4-10 mS/cm) may have a large impact on yield ranging from 21 to 100% while the reduction of high molecular weight is more subtle (0.0 to 0.45%). Charge heterogeneity data indicate that within this range of pH and conductivity the acidic charge species can be modulated greatly. Practically, the acidic charge variants can be reduce from as little as 3% to as much as 8%. The impact of mass loading on the resin also plays a role in the level of reduction that can be seen. Mass loading ranges tested included 30 to 60 g/L and showed a range of acidic charge variant reduction from 1% to 11% The development and data described above regarding the polishing chromatography step maps out a design space that shows the impact pH and conductivity have on process (yield) and product parameters (acidic species and HMW). Table 26 shows the preferred mobile phase conditions for the polishing chromatography of adalimumab. The contour maps shown above are indicative of a process that is robust and will produce adalimumab with the required purity (Table 27).

Table 26. CAPTO ^® SP ImpRes Polishing Chromatography Step

Table 27. CAPTO ^® SP ImpRes Eluate Impurity Profile

Eluate

Example 5

Nanofiltration

Nanofiltration is a requirement in the production of small and large proteins when using mammalian cell culture. Evaluation of the nanofiltration process was conducted varying protein concentrations as well concentrations of sodium chloride and their impact on flux and capacity. The results are shown in Table 28.

Table 28. Nanofiltration Parameters

The conditions selected are shown below in Table 29 and were tested in duplicate in a viral spiking study performed by a third party vendor to assess the LRV. The viral spiking study was performed with replicates of two model viruses. The data from the spiking study is shown in Table 30.

Table 29. Final adalimumab Nanofiltration Parameters

Table 30. Viral Spiking Study Results for adalimumab Nanofiltration

Example 6

Concentration and Diafiltration

The final step in the purification or downstream manufacturing process is the ultrafiltration or concentration and diafiltration of the product pool. This unit operation brings the product to its specified protein concentration during the ultrafiltration portion and buffer exchanged into the formulation vehicle that provides maximum product stability.

A polyethersulfone cassette with a 30 kDa molecular weight cutoff was evaluated for its performance with respect to the impact of TMP on permeate flux and product retention during ultrafiltration (UF) as well as performance on buffer exchange and contaminant removal during diafiltration. Ultrafiltration experiments were conducted bracketing a range of 10 - 65 g/L including process flux versus both transmembrane pressure (TMP) and time (Figures 14 and 15). Feed flow was tested from 300-500 L/m2/hr during UF experiments at both the initial product concentration (10 g/L) as well as the final concentration (60 g/L). TMP was evaluated with respect to time and process flux in the range of 2 - 30 psi. It was concluded that the optimum conditions to operate the UF portion of the step were with a feed flow rate of 450 L/m2/hr and a TMP of 10 psi. The membrane was challenged to a ratio of 500 g of adalimumab/m2 of filter area (Figure 16).

Diafiltration (DF) of adalimumab was evaluated by examining pH and conductivity changes in the permeate effluent over a range of diavolumes exchanges (0-10 diavolumes) as well as changes in process flux over time. The initial pH and conductivity of the DF starting material were 6.2 and 12.9 mS/cm respectively while the target pH and conductivity of the formulation vehicle was 5.2 and 4.0 mS/cm. Following only four diavolume exchanges the pH and conductivity of the material were 3.99 and 4.0 mS/cm. Process impurity levels such as Chinese Hamster Ovary host cell proteins, residual DNA and residual leached Protein A levels were also evaluated for additional clearance. Only residual DNA levels were reduced beyond initial levels. While the diafiltration is essentially complete following four diavolumes exchanges, the process may utilize five diavolumes to ensure complete diafiltration of adalimumab. Table 31 represents the impurity profile resulting from the complete adalimumab downstream process (the bulk drug substance).

Table 31. Bulk Drug Substance Impurity Profile Cone. Step

HCP DNA rPA (g/L) %HMW %MON %LMW %Acidic %Main %Basic Yield

(ppm) (ppb) (ppm)

(%)

Clarified

Unprocessed 0.56 1.0 97.0 2.0 24.9 55.0 20.1 2.3xl0 ⁵ 2.8xl0 ⁶ NA NA Bulk

MABSELECT SURE ^® 21.0 1.6 98.1 0.3 25.4 55.1 19.5 663 4193 0.48 97.0 Eluate

CAPTO

Adhere

5.44 0.3 99.4 0.3 21.3 57.0 21.7 130 3.5 <0.04 74.0 Flow- through

CAPTO SP

ImpRes 10.7 0.4 99.6 0.0 14.2 61.3 24.5 <1.1 <0.9 <0.02 70.0 Eluate

Bulk Drug

50.0 0.4 99.6 0.0 14.3 59.7 26.0 1.0 <0.9 <0.02 98.0 Substance

Example 7

Summary

Results to date are indicative of a purification process that is robust and capable of reducing even the most subtle product related impurities (acidic species) resulting in adalimumab bulk drug substance that is highly similar to the HUMI RA ^® (Abbvie

Biotechnology Ltd. Corp.) adalimumab reference product.

Example 8

Polishing Purification - Basic Species

The cation exchange chromatography in this Example is performed using a resin with a sulfonate or sulfopropyl chemistry. A series of experiments were performed to determine the most effective pH and conductivity to bind and elute adalimumab. The initial screening process is outlined in Table 32. The step as outlined and described here is primarily for the reduction of basic charge variants, leached Protein A, host cell proteins and potential viruses. The step is run in a bind and elute mode, and has a dynamic binding capacity of 30- 70 g antibody per liter of packed resin. Cation exchange chromatography is used primarily to reduce the remaining basic charge species to within range of the reference product (HUMIRA ^® adalimumab). Additional clearance of process and product related impurities (HMW, HCP, rPA, rDNA) as well as potential viruses can also be achieved during this step.

Table 32. I nitial cation exchange chromatography pH conditions screening process

Chromatography Step Parameter

Equilibration 40mM sodium phosphate, pH 5.8

Sample Loading adalimumab (Residence time = 2.5min) Wash 40mM sodium phosphate, pH 6.5

40mM sodium phosphate, conductivity gradient (0.025 - 0.5M

Elution

sodium chloride), pH 5.8

Strip 1M sodium chloride

Re-equilibration 40mM sodium phosphate, pH 5.8

Material was loaded at an acidic pH of 5.0. The pH was gradually increased in increments of 0.5 units and the elution profile was monitored at each point. Binding of adalimumab to the cation exchange support was disrupted at pH 7.0. The conductivity inherent to the mobile phase at pH 7.0 is 4.7 mS/cm. No clear separation of monoclonal antibody species is evident when only the pH is changed.

Additional experiments were conducted to modify the conductivity of the mobile phase to increase the resolution of the chromatography step. An initial experiment conducted at a pH of 5.5 to promote strong affinity and binding was carried out using a conductivity range of 90-150 mS/cm in three chromatography runs. The chromatograms indicate no improvement in resolution as result of the conductivity changes alone. This is due most likely to the charge interaction of the monoclonal antibody to the resin being too strong.

A follow up experiment was run at a pH of 6.5 and at the same conductivities as previously tested. At 90 mM sodium chloride, a shoulder on the descending slope of the UV absorbance profile emerged. This new shoulder is evidence of a separation of charged species in an early stage of resolution.

In order to continue improving the resolution of the chromatography, further optimization of the pH of the binding buffer is required. Reduction of the ionic strength of the buffer from 40 mM to 25 mM as well as a small increase in the pH (6.5 to 6.8) to reduce the affinity of the different charge species of the antibody slightly was attempted. A significant improvement in the resolution of the separation was observed, where a second peak was evident.

This set of experiments outlines a chromatography step using cation exchange chromatography that allows separation of basic charge variants from the predominant charged form of the adalimumab monoclonal antibody. Processing conditions are in Table 33 below. Table 34 shows the stepwise clearance that is achieved through the downstream process. Table 33. Cation exchange chromatography mobile phase strength processing conditions

Table 34. Cation exchange eluate impurity profile

Example 9

Effects of Varying Mass Load on Intermediate Purification The impact of varying mass load on the recovery and impurity clearance was evaluated. A study was performed under a range of binding capacities in which the flow through was collected and assessed based on product recovery, and acidic charge variant and high molecular weight species clearance. Table 35 shows the data from a mass loading study where the adalimumab Protein A eluate loaded onto the column varied from 50 g/L to 125 g/L. The relationship between mass loading and impurity clearances are shown in Figures 18-21.

The content of impurities that the feed-stream may have going on to the mixed mode resin and the clearance of any one or more the impurities that is required should dictate what mass loading is chosen. Hence, this provides a flexible chromatographic method for maintaining the desired impurity profile when the feed-stream may be variable. Table 35. CAPTO ^® Adhere™ HEPES buffer impact on mass load vs. impurities clearances.

Example 10

Effects of Varying Mass Load on Polishing Purification

The impact of mass loading on the resin also plays a role in the level of reduction that can be seen. A DoE was executed to map the design space when mass loading and conductivity are varied (pH is constant at 6.2). Table 36 shows the design and data from the study. Mass loading was tested from 30 to 60 g/L and showed a range of acidic charge variant reduction from 1 to 11% when the conductivity is varied by only from 5-7 mS/cm. Table 36. CAPTO ^® SP ImpRes mass loading DoE Loading Yield (%) AHMW AACV

Run # Cond (mS/cm)

(g/L)

1 30 5 97.4 0.10 -0.3

2 30 6 42.4 0.20 -8.5

3 30 7 15.6 0.60 -10.7

4 45 5 55.2 0.10 -6.9

5 45 6 35.5 0.20 -8.4

6 45 6 38.3 0.20 -6.9

7 45 7 16.7 0.60 -8.5

8 60 5 43.8 0.20 -7.3

9 60 6 20.0 0.70 -9.5

10 60 7 11.6 0.60 -9.9

The development was focused on understanding the Wash-2 cutting/transition criteria. A correlation between mass loading and Wash-2 peak cutting was derived based on previous experiments varying mass loading on the CAPTO ^® SP ImpRes (Figure 22). The line slope equation was used to predict the Wash-2 duration when initiated at 100 mAU on the ascending portion of the peak during the wash. The prediction was then tested using varied loading capacities and yield, HMW and acidic charge variant changes were monitored.

The data for mass loadings 30, 40 and 50 g/L are shown in Table 37, Table 38, and Table 39, respectively. The experiments described above were conducted at a scale suitable for screening (0.5cm x 10cm column) and were subsequently repeated at a scale

representative of manufacturing (1.6cm x 20cm). The data showed a reduced amount of acidic charge variant clearance upon scale up. The reduced amount of clearance was subsequently tested at the pilot scale (10cm x 20cm) with the same results. In order to provide a Wash-2 operation that would be flexible to a variable amount of acidic charge variants that may be present in the feed stock while maintaining an acceptable product recovery, a summary table (Table 40) was created from a series of chromatography runs (loaded to 30 g/L) at a column scale of 1.6cm x 20cm. The data can be used by

manufacturing to guide the operation of the polishing step as there may be variable levels of charge heterogeneity emanating from the bioreactor, which in turn requires different amounts of clearance from the CAPTO ^® SP ImpRes chromatography. Table 37. I mpact of peak cutting on HMW and acidic charge variant clearance at 30 g/L loading.

Table 38. I mpact of peak cutting on HMW and acidic charge variant clearance at 40 g/L loading.

Table 39. I mpact of peak cutting on HMW and acidic charge variant clearance at 50 g/L loading.

Table 40. CAPTO ^® SP I mpRes Wash-2 peak cutting summary (30 g/L Loading). Wash-2 Duration % ACV %HMW %ONS-3010

(CV) Removed Removed Recovery

0.16 2.50 0.19 93.6

0.32 2.70 0.16 91.7

0.65 4.00 0.11 88.1

0.97 4.10 0.12 83.3

1.30 5.40 0.14 79.6

1.62 5.90 0.16 76.7

90% of Wash- 2 Peak

5.9-7.5 NA >60.0

Max

The invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.