Monoclonal antibodies

MAb Contaminant Removal with a Multimodal Anion Exchanger A Platform Step to Follow Protein A Kjell Eriksson, Anders Ljunglöf, Gustav Rodrigo, and Eggert Brekkan Reprinted with permission from BioProcess International 7(2) (February 2009) Monoclonal antibodies (MAbs) constitute ~30% of the biopharmaceutical products currently under development (1). An increasing demand for MAbs during the past decade has led to intense development of highexpression cell cultures (2). Today, it is possible to see titers of 4 5 g/l, and expression levels as high as 15 g/l and greater have been reported. As a consequence, demand has increased for more efficient downstream processes. That demand, combined with its potential for reducing time-to-market, has increased interest in the value of platform approaches to MAb production (upstream as well as downstream). Many MAb producers are now adopting such approaches. Today, nearly all approved MAb processes include a capture step using protein A. In generic protocols with Pr o d u c t Fo c u s : Monoclonal antibodies Pr o c e s s Fo c u s : Downstream processing Wh o Sh o u l d Re a d: Process development, manufacturing Ke y w o r d s : HCP, multimodal chromatography, platform technologies, aggregates Le v e l: Intermediate direct capture of MAb from clarified cell culture supernatant, the high selectivity of protein A resins provides a high yield of very pure product. Protein A thus forms the foundation for the success of most MAb platform approaches to downstream processing. With the recently introduced Capto adhere multimodal anion-exchange resin, which has high selectivity for contaminants that remain after protein A separation, an additional step has been taken toward developing highly effective MAb downstream processes (3). The high purity that follows capture on protein A resins, combined with the multimodal functionality of Capto adhere resin, provides for highly productive, two-step processes (4, 5). MAb Platform Toolbox A platform process includes a number of unit operations, methods, and conditions based on experience gained from purifying a class of molecules that have similar properties ( 10). With >20 approved products and >10 in clinical trials, MAbs are just such a class of molecule (1). And the cornerstone for all MAb platforms is the unique selectivity of protein A. A platform technology enables rapid and economical process development and scale-up. By facilitating shorter development times, it potentially helps a greater number of drug candidates pass through development laboratories. The GE Healthcare (www.gelifesciences.com) use of highly similar platform approaches can also lead to greater robustness and smoother process transfer from development to manufacturing. Because MAbs are similar but not identical, their platforms cannot be completely fixed in all details; they must be flexible. The chromatography steps that follow a protein A step might, therefore, shift in order or be slightly different from process to process. A toolbox concept (Figure 1) could therefore be used to establish an efficient MAb purification process. For optimal results, it is most 52 BioProcess International February 2009

Figure 1: MAb chromatography resin toolbox *WO 2004/45, patent application from Lonza, may be relevant to a process that includes protein A followed by anion-exchange chromatography. Figures 1 7 @200 GE Healthcare, reproduced with permission. Cell Culture Cell Removal Figure 2: The Capto adhere ligand n-benzyln-methyl-ethanolamine has the potential for several types of interactions, the most pronounced being ion interaction, hydrogen bonding, and hydrophobic interaction. OH OH O O N + OH MabSelect SuRe Virus Inactivation and Filtration SP Sepharose FF/Capto S Pool for Final Filtration Ultrafiltration/Diafiltration important to optimize every step in a MAb process, including capture with protein A. Our suggested platform includes MabSelect SuRe protein A resin for capture, although MAbSelect and MabSelect Xtra brands also work very well for MAb platforms. The MabSelect SuRe product contains an alkalistabilized, protein A derived ligand that can be used with 0.1 0.5 M NaOH as a clean-in-place (CIP) agent, thereby prolonging its working life over that of other protein A resins (11, 12). The ligand is more stable in regards to proteolysis than a regular (native or recombinant) protein A ligand, which further reduces ligand leakage. In addition, its lack of Fab binding allows for more generic elution conditions such as (13) for a broad range of MAbs and Fc fusion proteins. All that facilitates the platform 0.2 µm Sterile Filtration Capto Q approach. The unique selectivity achievable with protein A media such as MabSelect SuRe brand makes a twochromatography step process possible. Such processes have been reported (14, 15). A Mu l t i m o d a l Anion Exchanger GE Healthcare s Capto adhere anion exchanger offers multimodal functionality designed for post protein A polishing in MAb processes. Contaminants left in a product pool after protein A capture are removed by operating Capto adhere resin in flowthrough mode so that they bind to the resin. Figure 2 shows the Capto adhere ligand: n-benzyl-n-methyl ethanolamine. This ligand displays several different modes of interaction, the most dominant being ionic interaction. However, other types of interaction such as hydrogen bonding and hydrophobic interaction also contribute. Multimodal anion exchangers have different selectivities from those of traditional strong anion exchangers (e.g., Q-types). Figure 3 compares descending gradient elution in bind elute mode of five different MAbs from Capto adhere and Capto Q resins. The order of elution is the same on both, but the at which the different MAbs elute is considerably lower with the Capto adhere product. This indicates that it involves additional interactions and that the multimodal functionality results in different selectivity. Capto adhere resin was initially designed for MAb purification, and its successful use in purification of an Fc-fusion protein has recently been reported (1). Nevertheless, this does not exclude using the multimodal anion exchanger in other applications such as the purification of other types of recombinant proteins. Op t i m i z at i o n o f Co n d i t i o n s Capto adhere resin is used following protein A in MAb purification processes. Its use to remove remaining contaminants is preferably done in flowthrough mode under conditions that Table 1: Overview of contaminant removal that can be expected with Capto adhere resin following protein A capture (MVM = minute virus of mice; MuLV = murine leukemia virus; LRV = log reduction factor) Contaminant/Step Protein A Eluate LRF: Capto adhere Contaminant Clearance HCP 500 10,000 ppm 2 3 Protein A 5 10 ppm 2 3 DNA 10 1,000 ppb 3 4 Viruses MVM MuLV N/A 5.5.5 3.5 5.0 Aggregated MAb 1 20% 1 2 Table 2: Virus removal efficiency of the Capto adhere multimodal anion exchanger; minute virus of mice (MVM) and murine leukemia virus (MuLV) were applied in flow-through mode with IgG MAb at.75 (LRF = log reduction factor at 95% confidence limit). Virus Conductivity (ms/cm) LRF MVM 10 >5. MVM 30 >5.9 MuLV 10 4.5±0.4 MuLV 30 3.±0.4 Fe b r u a r y 2009 BioProcess International 53

Figure 3: Comparing elution of five different antibodies on Capto adhere and Capto Q resins. Antibodies elute in the same order on both but much earlier (at a higher ) on Capto Q. The two were eluted using different gradient ranges (.2 4.9 for Capto adhere resin and 9.9.2 for Capto Q resin). 7 5 4 MAb 4:.22 MAb 3: 5.25 MAb 2: 5.12 MAb 1: 4.93 MAb 5: 5.14 10 20 30 40 ml allow antibodies to pass directly through a column while contaminants are adsorbed. In flow-through mode, loading conditions will be a compromise between those favoring yield and those favoring contaminant clearance. So loading conditions need to be optimized, and there will be a trade-off between yield and purity. Optimization is preferably done using design of experiments (DoE). When optimizing Capto adhere loading conditions, we have found that load,, and conductivity are the most 11 10 9 7 Capto Q MAb 3, 9.5 MAb 4, 9. MAb 5, 9.41 MAb 2,.37 MAb 1,.24 0 10 20 30 40 50 0 ml important factors. Optimizing them will influence both yield and clearance of contaminants. In DoE, all factors are varied simultaneously in a structured approach. A common method is to define a reference experiment (center point) and then perform other experiments around that point. DoE Example: The starting material was clarified cell culture supernatant containing monoclonal IgG1. Protein A chromatography used for the capture step was followed by viral inactivation at low. Polishing was then performed Figure 5: Example of response surfaces for yield and host-cell protein (HCP), dimers/aggregates (D/A), and leached protein A clearance Yield Conductivity 15 ms/cm Host Cell Protein Load 300 mg/ml Figure 4: Full factorial design for optimizing load conditions (sample load varied 75 300 mg/ml,, and conductivity 2 15 ms/cm) 2 75 Load 300 15 Cond. on Capto adhere resin in flow-through mode. A full-factorial design in three variables (load,, and conductivity) with three center points was set up to investigate the effects on yield and clearance of key contaminants: host cell proteins (HCP), dimers/aggregates (D/A), and leached protein A (Figure 4). Separate models were created for yield, clearance of HCP, D/A, and leached protein A (Figure 5). The response surfaces show how load,, and conductivity influence the different responses and how to reach desired values for each of them. Sweet-spot analysis was performed with the following criteria for acceptance: With a load 100 g/l resin, yield in flowthrough should be 90%, HCP 50 ppm, D/A 1%, and leached protein A 5 ppm. The sweet-spot (Figure ) is a point (red) at which the goals for yield and contaminant clearance are fulfilled. Because the goals for HCP and protein A removal are fulfilled in the entire experimental space, the optimization in this particular example was done between yield and D/A removal. Dimer/Aggregates Conductivity 15 ms/cm Protein A Load 300 mg/ml Figure : Sweet-spot analysis including all experimental data from the experimental design Load 300 20 20 240 220 200 10 10 140 120 100.0.5 7.0 7.5.0 54 BioProcess International Fe b r u a r y 2009

Op e r at i o n Capto adhere separation is operated so that antibodies pass directly through a column while contaminants are adsorbed. One contaminant that needs to be reduced/removed is the MAb itself in aggregated form. Using Capto adhere resin reduces not only the amount of aggregates, but also other contaminants of concern such as HCP, DNA, protein A, and viruses. Table 1 overviews the post protein A contaminant removal that can be expected using this multimodal anion exchanger. It is of particular interest to establish virus-removal efficiency. A study was thus performed to investigate removal of minute virus of mice (MVM) and murine leukemia virus (MuLV). Table 2 shows the results. Both model viruses are effectively removed at higher and lower ionic strengths. For a traditional anion exchanger such as a Q-resin, good virus clearance would not be expected at the higher ionic strength. Combining a protein A resin s high selectivity with the unique contaminantremoving properties of the Capto adhere anion exchanger makes it possible to cut many purification processes from three chromatography steps to two. To achieve this in practice, it is of utmost importance to optimize not only the multimodal anion-exchange step, but also the initial capture step. The newly developed MabSelect SuRe resin, with its alkaline-stable ligand that allows CIP with NaOH, is an obvious choice for this purpose. Key advantages are its proven long working life, high dynamic binding capacity, and low ligand leakage (11, 12, 17, 1). To further optimize the capture step, in some cases it may be necessary to develop an intermediate wash step that removes loosely bound contaminants before elution (19). Figure 7 shows one example of a two-step purification process for a MAb. The starting material was clarified Chinese hamster ovary (CHO) cell supernatant spiked with host cell proteins (HCPs). Capture was performed using MabSelect SuRe protein A resin. After sample application, an intermediate wash was run with 20 mm sodium phosphate, 5% isopropanol, and 0.5 M NaCl at 7.0. Analysis of the wash fraction showed that it mainly contained aggregates and HCP (19). MAbs were then eluted with sodium citrate 3.4. The HCP concentration was reduced from 130,000 ppm to 55 ppm, and the remaining aggregate content was 0.7%. MabSelect SuRe eluate was then purified further in flowthrough mode on Capto adhere anionexchange resin. As can be seen from the Table 3: In support of Figure 7 (HCP = host cell protein; D/A = dimers/aggregates; ND = not determined) Two-Step Process Yield (%) D/A (%) Protein A (ppm) HCP (ppm) Start material spiked with HCP 100 130,000 MabSelect SuRe 95 0.7 <1 55 Capto adhere 95 <0.1 ND 7.5 results, contaminant removal was excellent. HCP levels were reduced to <10 ppm, and both aggregate and leached protein A levels were below detection limits. A two-step chromatography process offers obvious advantages over a threestep process: Fewer steps improve yield and help shorten process times, translating to higher productivities (in grams produced over time). A two-step platform also improves process economy. In addition, less chromatography resin is used, less equipment and buffer volumes are needed, and the required floor space in a production facility can be reduced. When working on process optimization from productivity as well as process economy viewpoints, following a LEAN concept is highly advantageous. Reducing waste, materials, and especially time will lead to more efficient production (20). As described here, Capto adhere resin is usually run in flow-through mode, but in some cases it could be advantageous to use it in bind elute mode (1). In addition, some MAb preparations contain fragment levels that are too high for the final product. MAb fragments bind less strongly to this resin and could thus be separated from monomeric MAbs. Dimers and other aggregates bind more strongly and thereby elute after monomeric MAbs. Running this multimodal anion exchanger in bind elute mode thus has some advantages in certain situations. But it also has one major drawback: loading capacity. Loading in flow- Figure 7: Purification of a MAb from CHO cell culture (MabSelect SuRe capture step followed by Capto adhere resin in flow-through mode) A 20 nm (mau) 4,000 MAbSelect SuRe 10 A 20 nm (mau) 3,000 ms/cm 150 3,000 2,000 100 2,000 1,000 0 0 200 400 00 00 1,000 ml 4 1,000 50 0 0 5 10 15 20 25 ml Fe b r u a r y 2009 BioProcess International 55

through mode is often 100 300 g/l resin; in bind elute mode it will be in the range 25 40 g/l. However, the possibility of using Capto adhere resin in bind elute mode should make it an interesting alternative for cases in which its unique selectivity is required and also for proteins other than MAbs. Re f e r e n c e s 1 Roque ACA, et al. Antibodies and Genetically Engineered Related Molecules: Production and Purification. Biotechnol. Prog. 20(3) 2004: 39 54. 2 Birch JR, Racher AJ. Antibody Production. Adv. Drug Deliv. Rev. 5, 200: 71 5. 3 Johansson B-L, et al. Preparation and Characterization of Prototypes for Multi- Modal Separation Aimed for Capture of Positively Charged Biomolecules at High-Salt Conditions. J. Chromatogr. A 101(1) 2003: 35 49. 4 Johansson HJ, et al. Multi-Modal Chromatography for Purification of Monoclonal Antibodies (poster). Recovery of Biological Products XII 2 7 April 200, (Phoenix, AZ). American Chemical Society Biotechnology Division; www. recoveryconferences.org. 5 Eriksson K, et al. Post Protein A Removal of Contaminants from Monoclonal Antibodies with a Multi-Modal Anion Exchanger. 234th ACS National Meeting 19 23 August 2007 (Boston, MA). American Chemical Society; www.acs.org. Slaff G. Application of Technology Platforms to the Purification of Monoclonal Antibodies. BioProcess International European Conference 11 14 April 2005 (Berlin, Germany). IBC Life Sciences, www.informa-ls. com. 7 Sofer G, Chirica LC. Improving Productivity in Downstream Processing. BioPharm Int. 19, 200: 4 53. Shukla AA, et al. Downstream Processing of Monoclonal Antibodies: Application of Platform Approaches. J. Chromatogr. B 4, 2007: 2 39. 9 Ishihara T, Kadoya T. Accelerated Purification Process Development of Monoclonal Antibodies for Shortening Time to Clinic: Design and Case Study of Chromatography Processes. J. Chromatogr. A 117 (1 2) 2007: 149 15. 10 Low D, et al. Future of Antibody Purification. J. Chromatogr. B 4, 2007: 4 3. 11 Hahn R. Comparison of Protein A Affinity Sorbents III: Life Time Study. J. Chromatogr. A 1102, 200: 224 231. 12 Hober S, et al. Protein A Chromatography for Antibody Purification. J. Chromatogr. B 4, 2007: 40 47. 13 Ghose G, et al. Antibody Variable Region Interactions with Protein A: Implications for the Development of Generic Purification Processes. Biotechnol. Bioeng. 92() 2005: 5 73. 14 Vunnum S, et al. Anion Exchange Purification of Monoclonal Antibodies: Principles of Weak Partitioning Chromatography. 232nd ACS National Meeting 10 14 September 200 (San Francisco, CA); American Chemical Society, www.acs.org. 15 Kelley B. Very Large Scale Monoclonal Antibody Purification: The Case for Conventional Unit Operations. Biotechnol. Prog. 23(5) 2007: 995 100. 1 Rea DW, et al. Solutions for Purification of Fc-fusion Proteins. BioPharm Int. supplement, March 200: 20 25. 17 Johansson HJ. Advances in the Purification of Monoclonal Antibody Based Therapeutics. Antibody Development and Production 1 3 March 200 (Carlsbad, CA); IBC Life Sciences, www.ibclifesciences.com. 1 Jagschies G, et al. Technical and Economical Evaluation of Downstream Processing Options for Monoclonal Antibody (MAb) Production. BioPharm Int. supplement, June 200: 10 19. 19 Grönberg A, et al. A Strategy for Development of a MAb Purification Platform (poster). BioProcess International European Conference 20 23 February 200 (Prague, Czech Republic); IBC Life Sciences, www. informa-ls.com. Also BioProcess Int. 5(1) 2007: 4 54. 20 Jagschies G, et al. Modern Tools and LEAN Concepts for Manufacturing Flexibility. BioPharm Int. (in press). c Corresponding author Kjell Eriksson is a senior scientist in Life Sciences R&D at GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 4 Uppsala, Sweden; 4-1- 120539, kjell.eriksson@ge.com; www. gelifesciences.com. Anders Ljunglöf, Gustav Rodrigo, and Eggert Brekkan are all scientists at GE Healthcare. Capto, MabSelect, MabSelect SuRe, MabSelect Xtra and Sepharose are trademarks of GE Healthcare companies. Figures 1 7 200 GE Healthcare, reproduced with permission. 5 BioProcess International Fe b r u a r y 2009