WO2009091680A1 - High performance liquid chromatographic (hplc) purification of proteins using semi-compressible resins - Google Patents

Abstract

A process for the purification of a protein and, in particular a monoclonal antibody, is described which involves the use of a semi-compressible resin in a high performance liquid chromatography column operated at high flow rates and operating pressures. The process can be used for all stages of protein purification including protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography.

Description

TITLE OF THE INVENTION

HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC (HPLC) PURIFICATION OF

PROTEINS USING SEMI-COMPRESSIBLE RESINS

FIELD OF THE INVENTION

The invention relates generally to a method for the purification of proteins from a fermentation broth using rigid and semi-compressible high performance liquid chromatographic (HPLC) resins to produce a purified drug substance. The invention specifically provides a process for the purification of monoclonal antibodies.

BACKGROUND OF THE INVENTION

The large-scale, economic purification of proteins is increasingly an important problem for the biotechnology industry. Therapeutic proteins are typically produced by cell culture using either mammalian or bacterial cell lines engineered to express the protein of interest (POI) from a recombinant plasmid containing the gene encoding the protein. Separation of the expressed POI from the mixture of components needed to grow and maintain such cell lines and from any by-products of the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge to pharmaceutical manufacturers. The need for improved large scale processes for the purification of proteins has been heightened with the development of improved cell culture methods that result in an increased mass of product to be purified. Typical cell culture volumes were 5 to 10 kL in the 1990 time frame. Present day bioreactor volumes can now exceed 20 kL and some are as large as 30 kL. Titers for commercial products have typically been 1 to 2 g/L, but have recently increased to 3 to 5 g/L. Those of ordinary skill in the art are of the general belief that improvements in cell culture methods, combined with further progress in media and feed development, will result in typical antibody titers of at least 10 g/L (Birch and Archer, Advanced Drug Delivery Reviews, 59: 671 (2006); Kelley, Biotech Progress, 23: 995- 1008 (2007)).

To ensure the safety of such a human therapeutic, regulatory agencies, such as the Food and Drug Administration (FDA), have imposed stringent purity standards requiring that protein-based pharmaceutical products are substantially free from impurities, including product related contaminants, such as aggregates, fragments and variants of the recombinant protein, and process related contaminants, such as host cell proteins, media components, viruses, DNA and endotoxins. While various protein purification processes are utilized in the pharmaceutical industry, a typical purification process for an antibody includes an affinity-purification step, such as Protein A affinity chromatography, an anion exchange change (AEX) chromatography step and a cation exchange chromatography (CEX) step. Protein A affinity chromatography, the most commonly used primary capture step for proteins and, in particular, monoclonal antibodies (mAb), is where a mAb in a mixture primarily and selectively binds to protein A via its Fc region and the impurities, such as host cell proteins (HCPs), DNA and endotoxins, remain unbound (Lancet et al, Biochem, and Biophvs. Res. Comm., 85(2): 608-614 (1978); Sulica et al. Immunology, 38(1): 173-179 (1979); Ghose et al, Biotechnol Prog.. 20(3): 830-840 (2004); Ghose et al., Biotechnol Bioeng.. 96(4): 768-779 (2007)). However, this step does not routinely clear aggregates and it may add protein A into the mixture. AEX and CEX are used as complimentary polishing steps in the industry to remove DNA, endotoxin and any leached protein A ligand, as well as HCPs. CEX is also used to remove any aggregates in the product (Fahner et al., Biotechnol Genet. Eng, Rev.. 18: 301-327 (2001); Iyer et al. Bio. Pharm. Int. 15: 14-20 (2002); Shukla et al, J. Chrom. B, 848(1): 28-39 (2007)). While the available purification processes may achieve the desired purity standards, they often do so by a trade off in cost or productivity. Productivity limitations are typically imposed by the limited operating range, both in terms of the allowable pressures and flow rates, afforded by conventional chromatography resins. The resulting low productivity can result in long cycle times or high production costs. This problem has been solved in the past by increasing the scale of the chromatography purification to reduce the cycle time for the given step. For the early stages of product development (pre-clinical production) or early stage manufacture of drug compounds for safety assessment or Phase I clinical trials, where the chromatography resin is typically only used for a few cycles and which represents only a fraction of the demonstrated lifetime for the resin, this can result in a high unit cost for protein purification, i.e. dollars/gram of monoclonal antibody produced.

Applications of medium pressure chromatography have been described in the literature. For example, the use of a polystyrene divinyl benzene resin (POROS protein A) in a protein A purification has been described. Fahner et al. Bioprocess Engineering, 21 : 287-292 (1999), used such a method, but due to pressure limitations (of about 30 psi) of their system (acrylic and glass columns) it was necessary to reduce the length of their packed bed (bed height) in order to increase the linear flow rate to an acceptable level. The reduced bed height combined with the increased linear flow rate resulted in a significant reduction in the amount of POI that could bind to the resin (dynamic binding capacity). As shown herein, the use of HPLC provides improved packing and eliminates the need to reduce the height of the packed resin at an increased flow rate. This allows one to maintain, or optionally to increase, the dynamic binding capacity even at such an increased flow rate.

HPLC has been used for the purification of polypeptides and monoclonal antibodies. Santucci, A., et al., J. Immunological Methods, 114: 181-185 (1988), purified an antibody using the antigen as an immobilized ligand bound to the HPLC column matrix. Purified antibody is recovered by lowering the pH of the elution buffer. Pavlu, B., et al., J. Chromato graphy, 359: 449-460 (1986), purified an antibody in one chromatographic step by precipitating the antibody by ammonium sulphate prior to HPLC. Burchiel, S., et al., 1 Immunological Methods, 69: 33-42 (1984), purified a murine antibody utilizing anion exchange and gel permeation chromatograph using HPLC under neutral pH conditions with a hydrophilic resin.

Attempts have also been made to optimize HPLC to achieve acceptable product quality for a biological product. Nti-Gyabaah, J., et al., Biotechnol. Prog. 22: 538-546 (2006), restricted column loading in order to obtain the desired purity, albeit at limited resolution and productivity, of structural analogues of a pneumocandin. Roush, D., et al., J. Chromatography A, 827: 373-389 (1998), showed that yield and productivity are a function of, and that a tradeoff exists for the parameters, linear flow rate and column loading in their studies to optimize a normal-phase HPLC process for purification of fermentation derived echinocandins. Production scale processes using protein A purification of monoclonal antibodies has typically focused on optimizing flow rates and column length in order to design bioprocesses for maximum production (Fahrner, R., et al., Bioprocess Engineering, 21 : 287-292 (1999). Production scale processes have also used the performance characteristics of the sorbent to optimize productivity (Fahrner, R. et al., Biotechnol. Appl. Biochem., 30: 121-128 (1999), where differences in capacity and pressure drop affected the production rate.

Purification of polypeptides or antibodies have also been carried out using ion exchange chromatography utilizing multiple changes in the conductivity and/or pH of the buffers employed to separate the protein from the contaminants (U.S. Pat. No. 6,489,447). U.S. Pat. No. 6,797,814 describes a process for purifying an antibody by absorbing the antibody to protein A immobilized on a solid phase of silica or glass and then removing the contaminants bound to the solid phase with a hydrophobic electrolyte. Similarly, U.S. Pat. No. 5,641,870 describes purifying an antibody by loading the antibody onto a hydrophobic interaction chromatography column and eluting the product with a low pH buffer. Methods for the purification of a protein or antibody have also included the use of low temperature protein A affinity chromatography to reduce the leaching of protein A into the product mixture (U.S. Publ. Appln. 2005/0038231).

Applicants herein have developed a platform process utilizing HPLC and semi- compressible resins for all modes of purification typically employed for the manufacture of a therapeutic protein. The inventive method described herein is more cost effective and has improved productivity over prior methods employed therein.

SUMMARY OF THE INVENTION

In one embodiment the invention claimed herein is a process for purifying a protein of interest (POI) comprising:

(a) loading a liquid mixture, containing a POI at a concentration of about 200 mg/L to 5,000 mgL, at a linear flow rate in excess of 3,000 cm/h, and a column operating pressure of about 100 psi to 1,300 psi, onto about 20 cm to 30 cm packed bed height high performance liquid chromatography column packed with about 0.5L to 500L of a semi- compressible resin, thereby binding the POI in the liquid mixture to the semi -compressible resin;

(b) washing the column containing the bound POI with about 2 to 5 column volumes (CV) of a wash buffer to displace the liquid mixture;

(c) eluting the bound POI by washing the column with about 2 to 5 CV of an elution buffer; and (d) recovering the elution buffer containing the purified POI.

The process optionally includes an equilibration step prior to the loading of the liquid mixture containing the POL Steps (a) through (d) may be repeated following regeneration of the column with a regeneration buffer so as to completely utilize the resin life. In a preferred embodiment of the invention the column is stainless steel.

In another embodiment of the invention the claimed process is a protein A affinity chromatography step, wherein said semi-compressible resin is selected from the group consisting of a polystyrene/divinyl benzene resin or a silica based resin. The wash, elution, and regeneration of the column are carried out at a linear flow rate in excess of 720 cm/h, and the loading and elution of the liquid mixture containing the POI are carried out at a linear flow rate in excess of 720 cm/h.

In still another embodiment of the invention the claimed process is an anion exchange chromatography step, wherein the semi-compressible resin is selected from the group consisting of a polyvinyl aery lam ide resin and a polystyrene/divinyl benzene resin coupled to a quaternary amine Hgand. The wash, elution, and regeneration of the column are carried out at a linear flow rate in excess of 3,000 cm/h, and the loading of the liquid mixture containing the POI are carried out at a flow rate in excess of 1,200 cm/h, In yet another embodiment of the invention the claimed process is a cation exchange chromatography step, wherein the semi-compressible resin is selected from the group consisting of a polyvinyl acrylamide resin and a polystyrene/divinyl benzene resin coupled to a sulfopropyl based ligand. The wash, elution, and regeneration of the column are carried out at a flow rate of about 3,000 cm/h, and the loading of the liquid mixture containing the POI are carried out at a flow rate in excess of 1 ,200 cm/h.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the pressure/flow curve for a silica-Protein A resin (Asahi Glass SI-Tech, Kobe, Japan). Figure 2 depicts the pressure/flow curve for a POROS 5OA Protein A resin

(Applied Biosystems, Foster City, CA).

Figure 3 depicts the pressure/flow curve for a UNOsphere Q anion exchange resin (BioRad, Hercules, Pa),

Figure 4 depicts the pressure/flow curve for a POROS HQ50 anion exchange resin (Applied Biosystems, Foster City, CA).

Figure 5 depicts the pressure/flow curve for a UNOsphere S cation exchange resin BioRad, Hercules, CA).

Figure 6 depicts the pressure/flow curve for a POROS HS50 cation exchange resin (Applied Biosystems, Foster City, CA). Figure 7 depicts the yield (%) and productivity (kg POI/L-resin/hour) as a function of loading linear velocity for an Asahi silica-protein A resin (Asahi Glass SI-Tech, Kobe, Japan).

Figure 8 depicts the yield (%) and productivity (kg POI/L-resin/hour) as a function of loading linear velocity for a POROS 5OA protein A resin (Applied Biosystems, Foster City, CA).

Figure 9 depicts the yield (%) and productivity (kg POI/L-resin/hour) as a function of loading linear velocity for an UnoSphere Q AEX resin BioRad, Hercules, CA). Figure 10 depicts the yield (%) and productivity (kg POI/L-resin/hour) as a function of loading linear velocity for a POROS 50HQ AEX resin (Applied Biosystems, Foster City, CA).

Figure 1 1 depicts the yield (%) and productivity (kg POI/L-resin/hour) as a function of loading linear velocity for an UnoSphere S CEX resin (BioRad, Hercules, CA).

Figure 12 depicts the yield (%) and productivity (kg POI/L-resin/hour) as a function of loading linear velocity for a POROS HS50 CEX resin (Applied Biosystems, Foster City, CA).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term "protein" is used herein in the broadest sense and includes polypeptides made up of amino acid residues covalently linked together by peptide bonds.

The term "antibody" is used herein in the broadest sense and specifically refers to monoclonal antibodies or fragments thereof.

The term "medium pressure" or "moderate pressure" when referring to high performance liquid chromatography herein means a pressure of about 15 psi to 120 psi.

The term "high pressure" when referring to high performance liquid chromatography herein means a pressure of about 120 psi to 5000 psi. The term "high performance liquid chromatography column" herein refers to a column that can withstand of up to 1000 psi. For example, a non-ferrous column and, more specifically, a stainless steel column, such as LC.60.VE.900.70 column skid, Prochrom, Champigneulles, France, is a column of this type for purposes of the claimed invention.

The term "semi-compressible resin" as used herein refers to a mechanically stable resin, that is, one that does not irreversibly compress, collapse, or irreversibly yield to pressure stress of 200 psi (i.e. 200 pounds-force per square inch). This means that the resin does not permanently deform as a result of the application of 200 psi pressure. An example of a pressure flow curve for Capto Q₅ a commercial resin that exhibits this properly up to 50 psi, is presented in Wang, J. Chromatography A, 1155 (1): 74-84 (2007). The term "high salt buffer" as used herein refers to a buffer that contains salt in the range of about 0.2 to 1.0 M NaCl, see for example, Tugcu et al, Biotechnology and Bioengineering. 99(3): 599-613 (2008). The term "equilibration buffer" or "column conditioning buffer" as used herein refers to the buffer used to condition the column before the product is loaded onto the column, for example, a 25 mM sodium phosphate buffer used to condition the protein column before the feed is loaded can be an equilibration buffer, Tugcu et al._s Biotechnology_and Bioengineering, 99(3): 599-613 (2008).

The term "regeneration buffer" as used herein refers to the buffer used to clean the column to remove bound impurities, for example, a high salt buffer, a NaOH-containing, a detergent-containing, or a phosphoric acid-containing buffer, Tugcu et al., Biotechnology and Bioengineering. 99: 599-613 (2007). The term "column volume" or "CV" as used herein refers to the volume of packed resin inside the column including any void volume. For example, if a 10 L column is packed with 2 L of resin, one CV is 2 L.

The term "load flow rate" as used herein refers to the volumetric flow rate at which the protein of interest (POI) is loaded onto the column. The term "flow rate" or "linear flow rate" or "linear flow velocity (LV)" as used herein refers to the volumetric flow rate divided by the packed bed (or internal column) cross sectional area. For example, the linear flow rate for a column at a volumetric flow rate of 120 ml/hr (or 120 cm³/h) through a packed bed with a cross section area of 20 cm² would be as follows: 120 cm³/h = or 6 cm/h.

20 cm²

The term "operating pressure" as used herein is obtained by subtracting the column outlet pressure from the inlet pressure during pumping of fluid through a packed bed column (feed inlet pressure - feed outlet pressure = operating pressure). The term "productivity" as used herein refers to the amount of purified product, i.e. POI, recovered per quantity of chromatographic resin in a given time period, for example, kg POI purified/liter resin/hour.

The term "yield" as used herein is the of amount product recovered divided by the amount of product loaded into the column multiplied by 100. For example, a column loaded with a solution that contained 10Og of product, but from which 9Og of product was recovered from the elution stream, would have a 90% yield.

The phrase "HPLC platform process" as used herein refers to one or more steps, phases or modes, used to purify a protein of interest (POI), including protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography, carried out via HPLC.

The phrase "benchmark process" or "conventional process" or "prior art process" as used herein with respect to "protein A affinity chromatography" refers to one such as that of Kelley, B., BiotechnoL Prog.. 23: 995-1008 (2007) or Tugcu et al., Biotechnology and

Bioengineering. 99(3): 599-613 (2008), in which a glass column, packed with MabSelect™ (GE Healthcare, Piscataway, NJ), an industry standard for protein A affinity chromatography, is equilibrated with an equilibration buffer at a linear flow rate of 150-300 cm/h. Thereafter, the cell culture supernatant (product stream) is loaded onto the column using the same linear flow rate. Upon completion of the loading, the column is again washed with the equilibration buffer and the product is eluted. Because of column pressure limitations (maximum of 45 psi) and the compressibility of currently available agarose-based resins used in glass columns, the linear flow rate on the column herein can not exceed 300 cm/h.

The phrase "benchmark process" or "conventional process" or "prior art process" as used herein with respect to "anion exchange (AEX) chromatography" refers to a process such as that of Fahner et al._s Biotechnology and Genetic Engineering Reviews, 18: 301-327 (2001) or Tugcu et al., Biotechnology and Bioengineering, 99(3): 599-613 (2008), in which a quaternary amine ligand, coupled to a base matrix composed of agarose such as Q Sepharose Fast Flow (GE Healthcare, Piscataway, NJ) or acrylamido and vinylic monomers, such as UNOsphere Q (BioRad, Hercules, CA), is packed into a BPG glass column (GE Healthcare, Piscataway, NJ) and washed with a high salt buffer at 300 cm/h. The bed is then equilibrated an equilibration buffer and the product is loaded. The bed is again washed with the equilibration buffer, following which the bed is regenerated with NaOH. Because of column pressure limitations (maximum of 45 psi), the flow rate on the column for all steps herein cannot exceed 300 cm/h. The phrase "benchmark process" or "conventional process" as used herein with respect to "cation exchange (CEX) chromatography" refers to a process such as that of Fahner et al.. Biotechnology and Genetic Engineering Reviews. 18: 301-327 (2001) or Tugcu et al., Biotechnology and Bioengineering, 99(3): 599-613 (2008), in which a sulfopropyl-based ligand coupled to a base matrix composed of polystyrene-divinylbenzene such as, POROS^® HS50 (Applied Biosystems, Foster City, CA), or acrylamido and vinylic monomers, such as

UNOsphere S (BioRad, Hercules, CA), is packed into a glass column and washed with a high salt buffer at 300 cm/h. The bed is then equilibrated with an equilibration buffer and the product is loaded. The column is again washed with the equilibration buffer, eluted, and regenerated using NaOH. Because of column pressure limitations (maximum of 45 psi), the linear flow rate to the column for all steps herein cannot exceed 300 cm/h.

Processes for purification of proteins Typically a purification process is performed on a clarified harvested cell culture broth to ensure that the drug substance is suitable for a pharmaceutical formulation, i.e., that it contains low levels of aggregated product, host cell residuals (both DNA and host cell proteins), bioburden, and endotoxin. A protein purification process typically comprises three chromatographic stages that ultimately yield the final drug substance, including, affinity (protein A), cation exchange (CEX), and anion exchange (AEX). While Applicants have present them herein in a specific sequence, those of ordinary skill in the art would understand and know how to modify the sequence of the stages for each particular application.

The use of HPLC can provide advantages with respect to speed and resolution relative to other chromatographic systems. It allows the user to more accurately control the chromatographic conditions, which may result in a higher degree of reproducibility over a wide range of volumes and concentrations as compared to other forms of chromatography. HPLC can provide a relatively fast purification with a reduced dilution of recovered product compared to conventional methods. However, the conventional HPLC process has not been used on a large scale for each stage of a protein purification process because of pressure limitations imposed by available columns, which typically only allow use up to 7 atm or about 120 psi, and manufacturer recommended limits for available resins, that limit the pressure to below 3 atm or about 45 psi. Other disadvantages include the lack of commercially available preparative off-the-shell HPLC equipment, and high pressure column hardware.

The major problems associated with the use of conventional liquid chromatographic methods for large scale protein purification can be attributed to the types of column hardware and resins commercially available for use therein. Typical glass columns, such as BPG glass columns (GE Healthcare, Piscataway, NJ), being a moderate pressure column, have pressure limitations which dictate resin selection for use therein, i.e. selection is based on the recommended limits established by the vendor for any given resin. A typical resin for use in glass columns, such as MabSelect™, an agarose-based protein A resin (GE Healthcare,

Piscataway, NJ) has a recommended pressures of 45 psi or less. The conventional belief by those of ordinary skill in the art was that use outside the recommended operating pressures would result in the collapse or failure of the resin bed, with a resulting loss of velocity and productivity. As such, optimization of the conventional processes focused on feed loading flow rates, which were typically kept to a maximum of about 300 cm/h for a 20 cm packed resin bed height. Others attempted to maximize productivity of chromatography steps for the purification of monoclonal antibodies with available commercial resins by balancing the optimum amount of POI binding to the resin with an optimum linear flow rate during the feed loading step, Tugcu et al., Biotechnology and Bioengineering, 99: 599-613 (2007), Ghose et al, Biotechnology Progress. 20(3): 830-840 (2004). Without exception these efforts at optimization focused solely on the linear flow rate during the feed loading step. No attempts were made to optimize this parameter for any other stage of the purification process, nor were attempts made to optimize any cumulative impact in productivity by increasing the linear flow rate for the other steps of the process, such as the equilibration, elution, or regeneration steps.

Applicants herein have surprisingly found that semi-compressible resins can be employed with linear flow rates outside their conventionally accepted limits. As a direct result thereof, Applicants demonstrate for the first time that use of these resins outside the conventional range allows one to increase the linear flow rate, not only for feed loading, but throughout the purification process. As shown herein, significant increases in overall productivity were achieved that were more than additive for each individual stage of the purification process.

Thus, Applicants herein have developed a large scale platform process for purifying a protein and, in particular, an antibody, using HPLC for each stage of the purification process. The claimed process may use one or more steps to carry out the purification. As demonstrated herein, the inventive methods combine the use of a stainless steel HPLC column with a semi- compressible resin, such as a polyacrylamide or a polystyrene based resin, to produce a process that operates at higher pressures, with increased flow rates and reduced cycle times than conventional protein purification processes. While the specific methods herein utilize different semi-compressible resins, the use of a common type of resin allows for process efficiencies in the selection of columns and the use of a single pump skid/process platform, or single unit operation.

Qualification of Semi-Compressible Resins For purposes of evaluation only, the processes described herein were carried out using the anti-ADDL antibody as defined and described in a co-pending applications, US 2007/0081998, and PCT/US2006/040508, which published as WO 2006/055178. While the processes herein have been evaluated using an anti-ADDL antibody, those of ordinary skill in the art will appreciate and know how to modify the processes herein as appropriate for any protein or antibody that is subject to this type of purification.

The protocol for the evaluation of the protein A HPLC resins is set forth in Example IA. A summary description of the stationary phases evaluated and the characteristics of the protein A HPLC resins, including typical operating conditions utilized in the benchmark process, are provided in Table 1. In each instance, loading was evaluated at a fixed bed height (25 cm) and a fixed loading rate (3Og of product per liter of resin). In all cases, product recovery was comparable to that from the conventional process.

As shown in Figures 1 and 2, pressure-flow curves obtained by pumping a PBS buffer through the column at different flow rates and then monitoring the pressure drop across the column, both the silica protein A and POROS^® 5OA resins evaluated could withstand pressure of up to 1000 psi without collapsing or permanently deforming the resin. Based on limits suggested by the resin manufacturer, the maximum operating pressure tested was (45 psi) at a maximum flow rate of 300 cm/h for a 25 cm packed resin bed height. As a result of the pressure-flow experiments herein (Example IA), Applicants determined that semi-compressible resins could be used at the flow rates of the inventive process for a protein A HPLC process.

In a similar manner resins suitable for use in the AEX and CEX HPLC methods claimed herein were evaluated. Information for the AEX and CEX resins is provided in Tables 2 and 3, respectively. The protocols for the evaluation of the AEX and CEX HPLC resins are set forth in Example IB and 1C, respectively. As shown in Figures 3 and 4, pressure-flow curves measured with PBS for both the UNOsphere Q and the POROS^® HQ50 resins, respectively, indicated that the column could be operated at flow rates up to 5500 cm/h, corresponding to pressures greater than 200 psi, without permanent deformation of the resin. As a result of the pressure-flow experiments herein (Examples IB), Applicants determined that semi-compressible resins could be used at the flow rates of the inventive process for an AEX HPLC process.

Similarly, as shown in Figures 5 and 6, pressure-flow curves measured with PBS for both the UNOsphere S and the POROS^® HS50 resins, respectively, indicated that the column could be operated at flow rates up to 5500 cm/h, corresponding to pressures greater than 200 psi, without permanent deformation of the resin. As a result of the pressure-flow experiments herein (Examples 1C), Applicants determined that semi-compressible resins could be used at the flow rates of the inventive process for an AEX HPLC process.

Table 2

Table 3

Affinity (Protein A) Chromatography

The primary purification step for the production of high purity therapeutic proteins and, specifically monoclonal antibodies (mAb), is protein A affinity chromatography. Various approaches for increasing productivity for protein A affinity chromatography using medium pressure chromatography have been studied. See for example, the use of a polystyrene divinyl benzene resin (POROS protein A) in a protein A purification, Fahner et al., Bioprocess Engineering, 21: 287-292 (1999), where due to pressure limitations (of about 30 psi) of their system (acrylic and glass columns) it was necessary to reduce the length of their packed bed (bed height) in order to increase the linear flow rate to an acceptable level, Fahrner et al., Biotechnol. Appl. Biochem., 30: 121-128 (1999).

Due to pressure limitations of a medium pressure-rated column typically used in this type of chromatography, which use easily compress agarose-based resins as the column matrix, variations in the operating conditions, such as linear flow rate and column length, the use of a dual-linear flow rate loading strategy, either separately or combined with a continuous chromatographic purification have been proposed as ways to enhance productivity for protein A affinity chromatography. A disadvantage with these approaches is that higher flow rates in a relatively shorter column translate into a loss of resolution of impurities from the product of interest (dual axial and radial dispersion of impurities). Those of skill in the art have also proposed strategies of incorporating high throughput membrane adsorbers with immobilized protein A and novel ligands, Castilho et al., J. Mem. ScL 207 (2): 253-264 (2002). Disadvantages with this latter approach include inconsistent resolution of impurities, unacceptable product recovery, and the inability to regenerate the membrane for re-use. Still another approach for enhancing productivity of this method combines primary recovery with protein A via expanded bed chromatography, see for example, Thommes et al., J. Chromatography A, 752: 1 11-122 (1996) and Ohashi et al., Biotechnol. Prog., 18: 1292-1300 (2002), but major problems have been encountered with the fouling of the resin, which significantly impacts product quality.

In a typical protocol, such as that in Kelley, B., Biotechnol. Prog., 23: 995-1008 (2007) or Tugcu et al., Biotechnology and Bioengineering, 99(3): 599-613 (2008), a glass column, packed with MabSelect™, the industry standard for this type of purification, is equilibrated with 5 column volumes (CV) of an equilibration buffer at a linear flow rate of 150 to 300 cm/h using a 25 cm packed-resin column. Thereafter, the cell culture supernatant (product stream) is loaded onto the column using a linear flow rate of 150 to 300 cm/h. Upon completion of loading the feed, the column is washed with 5 column volumes (CV) of the equilibration buffer at 150 to 300 cm/h rate and the product is eluted at 150 to 300 cm/h, after which the column is regenerated with 5 column volumes (CV) of regeneration buffer at 150 to 300 cm/h.

Applicants herein have developed a method for protein A affinity chromatography that solves the aforementioned issues through the use of a semi-compressible resin in a high- performance-liquid-chromatography (HPLC) column. Applicants have found unexpectedly that semi-compressible resins, such as silica protein A (Asahi Glass SI-Tech, Kobe, Japan) or POROS^® 50A (Applied Biosystems, Foster City, CA) can be utilized outside conventional operating pressures and loading rates in an HPLC column to carry out protein A affinity chromatography without the loss of resin integrity, i.e. collapse, and resulting loss of flow rate. See Example 2. In a preferred embodiment of the invention, resins of this type are used in the claimed protein A HPLC method at flow rates of about 3,000 cm/h to 8,000 cm/h, corresponding to pressures from about 200 psi to 1 ,300 psi, more preferably at flow rates of about 4,000 cm/h to 7,000 cm/h, corresponding to pressures of about 600 psi to 1200 psi, and most preferably at flow rates of about 5,000 cm/h to 6,000 cm/h₅ corresponding to pressures of about 800 psi to 1000 psi.

The use of a semi-compressible resin circumvents the pressure limitations of the conventional protein A affinity chromatography methods, in the sense that the resin bed height was conserved and, in alternate embodiments was increased, while maintaining (or even increasing) the flow rate. Applicants' process herein resulted in increased speed, i.e. a faster linear flow rate without the pressure limits of the conventional process, with similar resolution of impurities. In some instances, the resolution of impurities can be enhanced as a result of reduced axial and radial dispersions.

The use of the inventive method allows one of ordinary skill in the art to more accurately control the chromatographic conditions, resulting in a higher degree of reproducibility over a wide range of volumes and concentrations, as compared to other forms of chromatography. The inventive method also provides a relatively fast purification with reduced dilution of recovered product as compared to other forms of chromatography, such as low or medium pressure chromatography.

Productivity for Protein A HPLC

Table 4A and 4B show the performance of the inventive HPLC method, using the Asahi silica-protein A and the POROS^® 50A resins, respectively, relative to a conventional protein A process. Yield and product quality were comparable to that achieved with the conventional process. Applicants have found unexpectedly that an HPLC process could be carried out for this stage of the protein purification and with linear flow rates significantly outside the conventional range, which resulted in an over ten-fold increase in overall productivity as compared to the conventional protein A process. As such, the increased linear flow rate would allow one to complete chromatographic purification of a batch about ten times faster than the conventional process.

Productivity Using Asahi Silica-Protein A Resin

The equilibration step was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the

5000 cm/h equilibration step was completed at a rate of about sixteen-fold faster = 16.7

300 cm/h

The loading step was carried out at a linear flow rate of 720 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the loading

720 cm/h step was completed at a rate of about thirteen-fold faster = 2.4

.300 cm/h

The chase wash was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the chase

5000 cm/h wash was completed at a rate of about sixteen-fold faster - 16.7

300 cm/h

The elution step was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the elution

5000 cm/h step was completed at a rate of about sixteen-fold faster 16.7

300 cm/h

The regeneration #1 step was carried out at a linear flow rate of 5_}000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that

5000 cm/h the chase wash step can be completed at a rate of about sixteen-fold faster = 16.7

300 cm/h

As compared to the conventional process, the cumulative impact of increasing the linear flow rates for all steps (including the loading step) for the inventive process translates to completion of the purification process for the protein A step in about four to five times faster than the conventional process. Table 4A

Productivity Using POROS^® 5OA Resin

The equilibration step was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the

5000 cm/h equilibration step was completed at a rate of about sϊxteen-fold faster = 16.7

300 cm/h

The loading step was carried out at a linear flow rate of 720 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the loading

720 cm/h step was completed at a rate of about thirteen-fold faster = 2.4

.300 cm/h

The chase wash was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the chase

5000 cm/h wash was completed at a rate of about sixteen-fold faster = 16.7

300 cm/h

The elution step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the elution

5000 cm/h step was completed at a rate of about sixteen-fold faster = 16.7

300 cm/h

The regeneration #1 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that

5000 cm/h the chase wash step can be completed at a rate of about sixteen-fold faster = 16.7

300 cm/h

As compared to the conventional process, the cumulative impact of increasing the linear flow rates for all steps (including the loading step) for the inventive process translates to completing the purification process for the protein A step in about four to five times faster than the conventional process.

Table 4B

Anion Exchange Chromatography

Even though significant purification of proteins and monoclonal antibodies can be achieved using a single protein A step, further clearance of residual HCPs₅ DNA, and other process related impurities is required to meet final purity specifications which can be achieved through the use of ion exchange resins, Fahrner et al., Biotechnology and Genetic Engineering Reviews, 18: 301-327 (2001). Among the ion exchange processes utilized, anion exchangers are typically used after a protein A step in a flow through mode for additional removal of DNA, HCPs₅ endotoxins and any leached protein A. Cation exchangers are employed for further reduction of the same contaminants plus the elimination of aggregates. To increase throughput for an ion exchange step, those skilled in the art have used shorter columns to allow faster flow rates without any pressure constraints. A disadvantage with this approach is that the use of higher flow rates in a relatively shorter column translates into a loss of resolution of impurities from the product of interest (dual axial and radial dispersion of impurities). Others skilled in the art of chromatography have incorporated high throughput membrane adsorbers, Castilho et al, Mem. ScL. 207(2): 253-264 (2002), Again, disadvantages with this approach have been inconsistent resolution of impurities, unacceptable product recovery, and the inability to regenerate the membrane for re-use. In a typical anion exchange (AEX) chromatography process, Fahner et al._s

Biotechnology and Genetic Engineering Reviews, 18: 301-327 (2001), carried out using a quaternary amine ligand, coupled to a base matrix composed of cross-linked agarose, such as Q Sepharose Fast Flow (GE Healthcare, Piscataway, NJ) or acrylamido and vinylic monomers, such as UNOsphere Q (BioRad, Hercules, CA)₅ is packed into a BPG glass column (GE Healthcare, Piscataway, NJ) and washed with 5 CV of high salt buffer at 300 cm/h. The bed is then equilibrated with 8 CV of equilibration buffer at the same flow rate. Product loading is carried out at a maximum of 300 cm/h. Upon completion of the loading, the bed is washed with 5 CV of equilibration buffer at the same flow rate, following which the bed is regenerated with 5 CV of NaOH at the same flow rate. Because of column pressure limitations (maximum of 45 psi), the linear flow rate has to be maintained at 300 cm/h or less for each of the elution steps. The system operates in a flow-through mode so that the product is not retained, which reduces the level of any exogenous DNA and HCP further.

To overcome the aforementioned issues. Applicants have developed a method for AEX that utilizes semi-compressible resins in high performance liquid chromatography (HPLC) columns operated in excess of 200 psi. Applicants have found unexpectedly that semi- compressible resins, such as UNOsphere Q (BioRad, Hercules, CA) and POROS^® HQ50 (Applied Biosytems, Foster City, CA) can be utilized outside conventional operating pressures and loading rates in an HPLC column to carry out an AEX chromatography without the loss of resin integrity. See Example 3. In a preferred embodiment of the invention, the resins were used in the claimed AEX HPLC method at flow rates of about 1 ,000 cm/h to 8,000 cm/h, corresponding to pressures from about 100 psi to 1,300 psi, more preferably at flow rate of about 4,000 cm/h to 8,000 cm/h, corresponding to pressures from about 150 psi to 1,300 psi, and most preferably at flow rates of about 5,000 cm/h to 7,000 cm/h, corresponding to pressures from about 180 psi to 1,150 psi. The flow rate of 5,000 cm/h was used for the equilibration, wash and regeneration steps, as compared to 450 cm/h in the conventional process, resulting in significantly faster flow rates (5,000 cm/h) overall, i.e. at each step of the process, and higher operating pressures (200 psi) than the conventional protocol. The use of a semi-compressible resin overcomes the pressure limitation of the conventional process such that the resin bed height could be the same as that of the conventional process or even be increased, while maintaining (or increasing) the flow rate. These operating conditions translate into increased speed, i.e. a faster linear flow rate without pressure concerns, with similar resolution of impurities as a result of reduced axial and radial dispersions.

Productivity of Anion Exchange HPLC

Tables 5 A and 5B shows a comparison of the operating conditions for the inventive AEX HPLC method using UNOsphere Q and POROS^® HQ50, respectively, relative to a convention AEX process. The use of the inventive method allows one of ordinary skill in the art to more accurately control the chromatographic conditions, resulting in a higher degree of reproducibility over a wide range of volumes and concentrations as compared to other forms of chromatography. The inventive method also provides a relatively fast purification with a reduced dilution of recovered product as compared to other forms of chromatography, such as low or medium pressure chromatography.

Productivity using UNOsphere Q resin

The equilibration step was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the

5000 cm/h equilibration step was completed at a rate of about eleven-fold faster = 1 1.1

450 cm/h

The loading step was carried out at a linear flow rate of 1 ,800 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the loading

1800 cm/h step was completed at a rate of about four-fold faster = 4

450 cm/h

The chase wash was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the chase wash

5000 cm/h was completed at a rate of about eleven-fold faster = 11.1

450 cm/h

The elution step was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the elution step

5000 cm/h was completed at a rate of about eleven-fold faster = 1 1.1

450 cm/h The regeneration # 1 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the

5000 cm/h chase wash step can be completed at a rate of about eleven-fold faster = 1 1.1

450 cm/h

The regeneration #2 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the

5000 cm/h chase wash step can be completed at a rate of about eleven-fold faster = 1 1.1

450 cm/h

As compared to the conventional process, the cumulative impact of increasing the linear flow rates for all steps (including the loading step) for the inventive process translates to completion of the purification process for the AEX step in about four to five times faster than the conventional process.

Table 5A

Productivity Using POROS® HQ50 resin

The equilibration step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the

5000 cm/h equilibration step was completed at a rate of about eleven-fold faster = 11.1 450 cm/h The loading step was carried out at a linear flow rate of 1800 cm/h (for the HPLC process - compared to 450 crn/h (for the conventional process). This means that the loading step

1800 cm/h was completed at a rate of about four-fold faster = 4

450 cm/h

The chase wash was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared Io 450 cm/h (for the conventional process). This means that the chase wash

5000 cm/h was completed at a rate of about eleven-fold faster = 1 1.1

450 cm/h

The elution step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the elution step

5000 cm/h was completed at a rate of about eleven-fold faster = 11.1

450 cm/h

The regeneration # 1 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the

5000 cm/h chase wash step can be completed at a rate of about eleven-fold faster = 11.1

450 cm/h

The regeneration #2 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the

5000 cm/h chase wash step can be completed at a rate of about eleven-fold faster = 11.1

450 cm/h

As compared to the conventional process, the cumulative impact of increasing the linear flow rates for all steps (including the loading step) for the inventive process translates to finishing up the purification process for the AEX step in about four to five times faster than the conventional process.

Table 5B

Cation Exchange Chromatography

In a conventional protein or monoclonal antibody purification process the refining purification is achieved through cation exchange (CEX) chromatography, which reduces levels of HCP and any other contaminants to pharmaceutically acceptable levels. The aforementioned issues and disadvantages associated with AEX processes are also concomitant with conventional CEX processes.

In a typical CEX protocol, Fahner et al._s Biotechnology and Genetic Engineering Reviews, 18: 301-327 (2001), a sulfopropyl-based ligand, coupled to a base matrix composed of polystyrene/divinylbenzene, such as POROS^® HS50 (Applied Biosystems, Foster City, CA), or acrylamido and vinylic monomers, such as UNOsphere S (BioRad, Hercules, CA) is packed into a glass column and washed with 5 CV of high salt buffer at a standard linear flow rate of 450 cm/h. Thereafter, the column is equilibrated with 8 CV of equilibration buffer. The product is then loaded at the standard flow rate. Following loading, the column is washed with 5 CV and eluted. Following product collection the column is regenerated using 5 CV of NaOH, Because of column pressure limitations (maximum of 45 psi), the linear flow rate has to be maintained at 450 cm/h or less for each elution step. Applicants herein have developed a method for a CEX chromatography that overcomes the aforementioned issues with ion exchange processes through the use of a semi- compressible resin in a high performance liquid chromatography (HPLC) column. Applicants have found unexpectedly that semi-compressible resins, such as UNOsphere S (BioRad, Hercules, CA) and POROS^® HS50 (Applied Biosystems, Foster City, CA) can be utilized outside conventional operating pressures and loading rates in an HPLC column to carry out a CEX chromatography. See Example 4. In a preferred embodiment of the invention, the resins were used in the claimed CEX HPLC method at flow rates of about 1,000 cm/h to 8,000 cm/h, corresponding to pressures from about 100 psi to 1,200 psi, more preferably at flow rate of about 4,000 cm/h to 8,000 cm/h, corresponding to pressures from about 150 psi to 1,200 psi, and most preferably at flow rates of about 5,000 cm/h to 6,000 cm/h, corresponding to pressures from about 180 psi to 1,000 psi. The flow rate of 5,000 cm/h was used for the equilibration, wash and regeneration steps, as compared to 450 cm/h in the conventional process, resulting in significantly faster flow rates (5,000 cm/h) overall, i.e. at each step of the process, and higher operating pressures (200 psi) than the conventional protocol.

Tables 6 A and 6B show a comparison of the operating conditions for the inventive CEX HPLC process using UNOshpere S (BioRad, Hercules, CA) and POROS^® HS50 (Applied Biosystems, Foster City, CA), respectively, relative to a conventional CEX process. The increased flow for the inventive process allows one to complete the chromatographic purification of a batch more than four times faster than the conventional process.

Productivity of UNOsphere S Resin

The equilibration step was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the

5000 cm/h equilibration step was completed at a rate of about eleven-fold faster = 11.1

450 cm/h

The loading step was carried out at a linear flow rate of 1,800 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the loading step

1800 cm/h was completed at a rate of about four-fold faster = 4.0 450 cm/h The chase wash was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the chase wash

5000 cm/h was completed at a rate of about eleven-fold faster = 11.1

450 cm/h

The elution step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the elution step

5000 cm/h was completed at a rate of about eleven-fold faster = 1 1.1

450 cm/h

The regeneration #1 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the

5000 cm/h chase wash step can be completed at a rate of about eleven-fold faster = 11.1

450 cm/h

The regeneration #2 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the

5000 cm/h chase wash step can be completed at a rate of about eleven-fold faster = 11.1

450 cm/h

As compared to the conventional process, the cumulative impact of increasing the linear flow rates for all steps (including the loading step) for the inventive process translates to completion of the purification process for the AEX step four to five times faster than the conventional process.

Table 6A

As a result of the experiments conducted herein, Applicants have found unexpectedly that an HPLC process could be carried out for a CEX stage of protein purification and with linear flow rates significantly outside the conventional range, which resulted in an over ten-fold increase in overall productivity as compared to the conventional CEX process. As such, the increased flow rates would allow one to complete chromatographic purification of a batch about ten times faster than the conventional process.

Productivity Using POROS^® HS resin

The equilibration step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the

5000 cm/h equilibration step was completed at a rate of about eleven-fold faster = 11.1 450 cm/h The loading step was carried out at a linear flow rate of 1800 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the loading step

1800 cm/h was completed at a rate of about four-fold faster = 4

^F 450 cm/h

The chase wash was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the chase wash

5000 cm/h was completed at a rate of about eleven-fold faster = 11.1

450 cm/h

The elution step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process ™ compared to 450 cm/h (for the conventional process). This means that the elution step

5000 cm/h was completed at a rate of about eleven-fold faster = 11.1

450 cm/h

The regeneration #1 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the

5000 cm/h chase wash step can be completed at a rate of about eleven-fold faster = 11.1

450 cm/h

The regeneration #2 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the

5000 cm/h chase wash step can be completed at a rate of about eleven-fold faster = 11.1

450 cm/h

As compared to the conventional process, the cumulative impact of increasing the linear flow rates for all steps (including the loading step) for the inventive process translates to completion of the purification process for the CEX step eight to nine times faster than the conventional process.

Impact of Increasing Linear Velocity for Loading on Yield

For the previous three chromatography steps (protein A, AEX and CEX) Applicants evaluated productivity at a fixed product loading, 30 g/L. To further describe and define the invention herein the impact of yield and productivity as a function of a range of increasing linear velocity for the loading step for all three chromatography steps was evaluated using an anti-ADDL antibody as described in US 7,241,444. While the range of linear velocity has been evaluated using the aforesaid anti-ADDL antibody, those of ordinary skill in the art will appreciate and know how to modify the processes as appropriate for any protein or antibody for which this type of purification is employed. Protein A Chromatography

The performance of the Asahi silica-protein A and POROS® 50A protein A resins were evaluated over a range of loading linear velocities from 720 to 2400 cm/h. A summary of the operating conditions and the associated yields are presented in Tables 7 A and 7B, respectively. As shown in Figures 7 and 8 for the Asahi and the POROS® resins, respectively, while the overall yield decreased as linear velocity increased for the loading step from 720 to 2400 cm/h, the overall productivity increased proportionally with increased linear velocity. For the Asahi resin, productivity reached a plateau at linear velocity of 1800 cm/h. For the POROS® 50A resin, productivity continued to increase up to the maximum linear velocity evaluated (2400 cm/h),

Table 7A

Table 7B

Flow rate Linear flow rate Pressure (mL/min) (cm/h) (psi)

Equilibration 22.2 8000 690

Chase wash 22.2 8000 650

Elution 22.2 8000 695

Regeneration 22.2 8000 718

Sanitization 0.8 300 85

20% EtOH 16.6 8000 854 storage mL/min cm/h Pressure (psi) Yield (%)

2.0 720 380 93

Loading 3.3 1200 450 85

5.0 1800 590 69

6.6 2400 690 58

Anion Exchange Chromatography

In a similar manner, the performance of the UNOsphere Q and the POROS® HQ50 AEX resins were evaluated over a range of loading linear velocities from 720 to 2400 cm/h. A summary of the operating conditions and the associated yields are presented in Tables 8 A and 8B, respectively. As shown in Figures 9 and 10 for the UNOsphere Q and the POROS HQ50 resins, respectively, while the yield is insensitive to the linear velocity in the range examined, productivity increased proportionally with increases in linear velocity for both resins.

Table 8A

Flow rate Linear flow rate Pressure (mL/min) (cm/h) (psi)

Pre- 16.6 6000 180 equilibration

Equilibration 16.6 6000 170

Chase / Wash 16.6 6000 180

Regeneration 16.6 6000 170

Pre-use 0.8 300 25 Sanitization

Post-use 0.8 300 25 Sanitization mL/min cm/h Pressure (psi) Yield (%)

2.0 720 50 94

Loading 3.3 1200 70 95

5.0 1800 110 93

6.6 2400 120 96

Table 8B

Cation Exchange Chromatography

The performance of theUNOsphere S and the POROS® HS50 CEX resins were also evaluated over a range of loading linear velocities from 720 to 2400 cm/h. A summary of the operating conditions and the associated yields are presented in Tables 9A and 9B_> respectively. As shown in Figures 11 and 12 for the UNOsphere S and POROS® HS50 CEX resins, respectively, while the overall yield decreased as linear velocity for the loading step increased from 720 to 2400 cm/h, the overall productivity continued to increase proportionally with increasing linear velocity. For UNOsphere S, a plateau was reached at 1800 cm/h while for POROS® HS50 productivity continued to increase up to 2400 cm/h. Table 9A

Flow rate Linear flow rate Pressure (mL/min) (crα/h) (psi)

Pre- 16.6 6000 210 equilibration

Equilibration 16,6 6000 195

Chase / Wash 16.6 6000 180

Regeneration 16.6 6000 200

Pre-use 0.8 300 35 Sanitization

Post-use 0.8 300 35 Sanitization mL/min cm/h Pressure (psi) Yield (%)

2.0 720 70 90

Loading 3.3 1200 90 75

5.0 1800 130 61

6.6 2400 145 49

Table 9B

Flow rate Linear flow rate Pressure (mL/min) (cm/h) (psi)

Pre- 22.2 8000 830 equilibration

Equilibration 22.2 8000 840

Chase / Wash 22.2 8000 810

Regeneration 22.2 8000 830

Pre-use 0.8 300 85 Sanitization

Post-use 0.8 300 85 Sanitization mL/min cm/h Pressure (psi) Yield (%)

2.0 720 380 94

Loading 3.3 1200 440 88

5.0 1800 520 72

6.6 2400 590 61

Platform Process for Protein Purification

A summary comparison of the operating conditions and results, in terms of overall cycle time and productivity, for the inventive processes herein relative to a conventional platform, i.e. a process incorporating all three stages of protein purification, is shown in Table 10. Data included in this table as to MabSelect SuRe™ (GE Healthcare, Piscataway_> NJ), an industry standard, is based on published results, see for example, Kelley et al., Biotechriol, Prog.. 23:995-1008 (2007) and Tugcu et al., Biotechnology and Bioengineering, 99(3): 599-613 (2008). Applicants have found that an HPLC process can be carried out for each stage of protein purification and that such processes can be carried using a semi-compressible resin in a stainless steel column significantly outside conventional process limits. The inventive platform process, unexpectedly resulted in significantly increased flow rates with corresponding significant reductions in overall cycle time and increases in productivity. In each stage, product quality either met or exceeded industry standards. As a result, the claimed process offers advantages relative to the conventional process by providing cost effective use of the resins, reduced cycle times,, increased productivity and an overall lower unit cost for protein purification.

As shown in Table 10, for the Protein A step (for both the Asahi silica-protein A and the POROS^® 5OA resins), using the increased linear flow rate of the inventive process for all steps, Applicants were able to complete seven runs for a typical batch in less than two hours, as compared to about 26 hours for the conventional process. This translates to more than five-fold increase in productivity (amount of POI purified per mass of resin per unit time).

Similarly, as shown in Table 10 (for the AEX step POROS^® HQ50, and the UNO Q resins), using the increased linear flow rate for all steps, Applicants were able to complete the purification in less than one hour, as compared to about four hours for the conventional process, This translates to more than five-fold increase in productivity (amount of POI purified per mass of resin per unit time).

Likewise, as shown in Table 10, for the CEX step (for the POROS^® HS50, and the UNO S resins), using the increased linear flow rate for all the steps, Applicants were able to complete two runs for a typical batch in about two hours, as compared to about nine hours for the conventional process. This translates to about a five-fold increase in productivity (amount of POI purified per mass of resin per unit time).

Combining all three chromatographic purifications, the inventive process allowed Applicants to complete the protein purification in about five hours, as compared to about 40 hours for the conventional process. This translates to about an eight-fold increase in productivity for the HPLC process.

Table 10

The following non-limiting examples, given by way of illustration, are demonstrative of the present invention. In addition to the definitions provided herein, the terms and abbreviations used herein are consistent with those used by those of skill in the art, including the following: anion exchange - AEX; cation exchange - CEX; centimeter - cm; centimeters per hour - crn/h or cm/hr; grams per liter - g/L or g/1; high performance liquid chromatography - HPLC; kiloliter - kL or kl; liter - L or 1; monoclonal antibody - niAb; phosphate buffer - PBS; pounds per square inch - psi; protein of interest - POL EXAMPLE 1 Qualification of Semi-Compressible Resins

In order to identify semi-compressible resins suitable for the HPLC methods herein, Applicants evaluated the mechanical stability of several protein A, AEX and CEX resins using phosphate buffer (PBS) to generate pressure flow curves.

A. Protein A Resins

High pressure columns were packed at 1,000 psi by Princeton Chromatography Inc., Cranbury, NJ, using the following resins: POROS 50A^® protein A (Applied Biosystems, Foster City, CA) and Asahi silica protein A resin (Asahi Glass SI-Tech, Kobe, Japan). Bed dimension for each column was 0.46 cm internal diameter x 25 cm length (column volume = 4.2 mL). The evaluation involved using a Waters FractionLynx™ HPLC System HPLC (Waters Corporation, Milford, MA) system to pump phosphate buffer (PBS) 10 mM sodium phosphate, 100 mM NaCl, pH 7.2 (Hyclone, Logan, UT) through each column at different flow rate and monitoring the pressures.

B. Anion Exchange Resins

High pressure columns were packed by Princeton Chromatography Inc., Cranbury, NJ, using the following resins: POROS^® HQ50 (Applied Biosystems, Foster City, CA) and UNOsphere Q (BioRad, Hercules, CA). Bed dimension for each column was 0.46 cm internal diameter x 25 cm length (column volume = 4.2 mL).

The evaluation involved using a Waters FractionLynx™ HPLC System HPLC (Waters Corporation, Milford, MA) system to pump phosphate buffer (PBS), 10 mM sodium phosphate, 100 mM NaCl, pH 7.2 (Hyclone, Logan, UT) through each column at different flow rate and monitoring the pressures. C. Cation Exchange Resins

High pressure columns were packed by Princeton Chromatography Inc., Cranbury, NJ, using the following resins: POROS^® HS50 (Applied Biosystems, Foster City, CA) and UNOsphere S (BioRad, Hercules, CA). Bed dimension for each column was 0.46 cm internal diameter x 25 cm length (column volume = 4.2 mL). The evaluation involved using a Waters FractionLynx HPLC™ System HPLC (Waters Corporation, Milford, MA) system to pump phosphate buffer (PBS) 10 mM sodium phosphate, 100 mM NaCl, pH 7.2 (Hyclone, Logan, UT) through each column at different flow rate and monitoring the pressures. EXAMPLE 2 Affinity Protein A HPLC

For purposes of demonstrating the processes herein, an anti-ADDL mAb (WO 2006/055178) was employed. Those of ordinary skill in the art would understand and know how to use these processes for other antibodies and proteins.

Phosphate buffer (PBS), 10 mM sodium phosphate, 100 mM NaCl, pH 7.2 were purchased from Hyclone, Logan, UT. Buffer components: citric acid and citrate trihydrate were purchased from Fisher Scientific (Pittsburg, PA) and sodium phosphate dibasic and monobasic, Tris-HCl, Trizmabase, and 5 M NaCl solution were purchased from Sigma (St. Louis, MO).

Analytical protein A HPLC analysis of samples was performed on an Agilent 1100 HPLC system (Agilent, Palo Alto, CA) equipped with a POROS PA ID immunoaffinity cartridge. Chromatography experiments were performed using Waters FractionLynx HPLC™ System (Waters Corporation, Milford, MA). DNA and host cell protein (HCP) levels in the product pools were tested using the Picogreen assay Guilloa et al., J. of Chromatography A, (1 113) Issues 1-2: 239-243 (2006).

For each run, either the Asahi silica protein A (Asahi Glass SI-Tech, Kobe, Japan) or POROS^® 5OA protein A (Applied Biosystems, Foster City, CA) column was equilibrated with 5 column volumes (CV) of PBS. Thereafter, sterile filtered mAb cell culture supernatant was loaded 30 g/L resin onto the column, after which it was washed with 5 CV of PBS, the product was eluted with citrate buffer, and then quenched with Trizma base. The column was regenerated with 3 CV of H₃PO₄ solution then sanitized with 5 5CV of NaOH solution. The product stream was analyzed for mAb concentration and impurity clearance. Specific flow rates and column operating pressures are shown in Tables 4A and 4B, for the Asahi silica protein A and the POROS^® 50A protein A resins, respectively.

EXAMPLE 3 Anion Exchange HPLC

As with the Protein A affinity HPLC (Example 2), an anti-ADDL mAb (WO 2006/055178) was employed to demonstrate the inventive process.

Phosphate buffer (PBS), 25 mM sodium phosphate, pH 7.5 were purchased from Hyclone, Logan, UT. 5 M NaCl solution were purchased from Sigma (St. Louis, MO). Analytical protein A HPLC analysis of samples was performed on an Agilent 1100 HPLC system (Agilent, Palo Alto, CA) equipped with a POROS PA ID immunoaffinity cartridge. Chromatography experiments were performed using Waters FractionLynx HPLC System. DNA and host cell protein (HCP) levels in the product pools were tested using the Picogreen assay Guilloa et al., J, of Chromatography A, (11 13) Issues 1-2: 239-243 (2006).

For each run, either a UNOsphere Q (BioRad, Hercules, CA) of a POROS^® 50HS (Applied Biosystems, Foster City, CA) column was equilibrated with 8 CV of PBS. Thereafter, sterile filtered product (from Example 2) was loaded 30 g/L resin onto the column. Product was collected as flow-through during the loading (product does not bind to the resin). Following loading, the column was washed with 5 CV of PBS. Bound impurity was then eluted with phosphate buffer (PBS), 25 mM sodium phosphate (pH 7.5) + IM NaCl. The column was regenerated with 8 CV of NaOH solution. The product stream was analyzed for niAb concentration and impurity clearance. The results were used to calculate the mass of product recovered. Specific flow rates and column operating pressures are shown in Tables 5A and 5B, for the UNOsphere Q and the POROS^® HQ50 columns, respectively.

EXAMPLE 4 Cation Exchange HPLC

As with the AEX HPLC (Example 3), an anti-ADDL mAb (WO 2006/055178) was employed to demonstrate the inventive process.

Phosphate buffer (PBS), 50 mM citrate buffer, pH 4.5 were purchased from Hyclone, Logan, UT. 5 M NaCl solution were purchased from Sigma (St. Louis, MO). Analytical protein A HPLC analysis of samples was performed on an Agilent 1 100 HPLC system (Agilent, Palo Alto, CA) equipped with a POROS PA ID immunoaffinity cartridge. Chromatography experiments were performed using Waters FractionLynx HPLC™ System (Waters Corporation, Milford, MA). DNA and host cell protein (HCP) levels in the product pools were tested using the Picogreen assay Guilloa et al., J. of Chromatography A (1 113) Issues 1-2: 239-243 (2006).

For each run, either the UNOsphere Q (BioRad, Hercules, CA) or the POROS^® 50HQ (Applied Biosystems, Foster City, CA) column was equilibrated with 8 CV of PBS. Thereafter, sterile filtered AEX product (from Example 3) was loaded 30 g/L resin onto the column. Following loading, the column was washed with 5 CV of PBS. Bound product was then eluted with phosphate buffer (PBS), 25 niM sodium phosphate (pH 4.5) + 0320 M NaCl. The column was regenerated with 8 CV of NaOH solution. The product stream was analyzed for mAb concentration and impurity clearance. The results were used to calculate the mass of product recovered. Specific flow rates and column operating pressures are shown in Tables 6A and 6B, for the UNOsphere S and the POROS^® HS50 columns, respectively.

Claims

WHAT IS CLAIMED IS:

1. A process for purifying a protein of interest (POI) comprising: a. loading a liquid mixture, containing a POI at a concentration of about 200 mg/L to 5,000 mg/L, at a linear flow rate in excess of 3000 cm/h and a column operating pressure of about 100 psi to 1,300 psi, onto about 20 cm to 30 cm packed bed height high performance liquid chromatography column packed with about 0.5 L to 500 L of a semi- compressible resin thereby binding the POI in the liquid mixture to the semi-compressibie resin; b. washing the column containing the bound POI with about 2 to 5 volumes of a wash buffer to displace the liquid mixture; c. eluting the bound POI by washing the column with about 2 to 5 volumes of an elution buffer; and d. recovering the elution buffer containing the purified POI.

2. The process of claim 1 wherein said high performance chromatography column is stainless steel.

3. The process of claim 1 wherein said POI is a monoclonal antibody.

4. The process of claim 1 wherein the process is a protein A affinity chromatography step and where said semi-compressible resin is selected from the group consisting of a polystyrene/divinyl benzene resin or a silica based resin.

5. The process of claim 4 wherein the wash, elution, and regeneration of the column are carried out at a linear flow rate in excess of 720 cm/h and the loading and elution of the liquid mixture containing the POI are carried out at a linear flow rate in excess of 720 cm/h.

6. The process of claim 1 wherein the process is an anion exchange chromatography step and where the semi-compressible resin is selected from the group consisting of a polyvinyl acrylamide resin and a polystyrene/divinyl benzene resin coupled to a quaternary amine ligand.

7. The process of claim 6 wherein the wash, elution, and regeneration of the column are carried out at a linear flow rate in excess of 3,000 cm/h and the loading of the liquid mixture containing the POI are carried out at a linear flow rate in excess of 1,200 cm/h.

8. The process of claim 1 wherein the process is a cation exchange chromatography step and where the semi-compressible resin is selected from the group consisting of a polyvinyl acrylamide resin and a polystyrene/divinyl benzene resin coupled to a sulfopropyl based ligand.

9. The process of claim 8 wherein the wash, elution, and regeneration of the column are carried out at a linear flow rate in excess of 3,000 cm/h and the loading of the liquid mixture containing the POI are carried out at a linear flow rate in excess of 1,200 cm/h.