Monoclonal antibodies

Transcription

1 V E N D O R Voice A Strategy for Developing a Monoclonal Antibody Purification Platform Anna Grönberg, Elin Monié, Maria Murby, Gustav Rodrigo, Elisabeth Wallby, and Hans J. Johansson Monoclonal antibodies (MAbs), the fastest growing segment within the biopharmaceutical industry, currently represent about 25% of the field (1). More than 20 monoclonal antibodies and Fc fusion protein pharmaceuticals are approved for sale in the United States and Europe (2), and approximately 200 MAbs are in clinical trials for a wide variety of indications (3). The market is predicted to continue to grow by 20.9% per year and reach US$16.7 billion in 2008 (4). The vast need for MAbs for preclinical and clinical studies justifies standardizing the production process, from cell culture to downstream purification, as well as related analytical tools. Platform technologies are standard sets of unit operations, conditions, and methods applied to molecules of a given class. They are used by many large PRODUCT FOCUS: MONOCLONAL ANTIBODIES PROCESS FOCUS: PURIFICATION, DOWNSTREAM PROCESSING WHO SHOULD READ: PROCESS DEVELOPMENT, PRODUCT DEVELOPMENT, AND MANUFACTURING KEYWORDS: MONOCLONAL ANTIBODIES (MABS), CHROMATOGRAPHY, PROTEIN A, PLATFORM PROCESS, OPTIMIZATION LEVEL: INTERMEDIATE biopharmaceutical companies and allow for rapid and economical product development from R&D through clinical phases to manufacturing (5). The platform approach is applicable to monoclonal antibodies because they represent a relatively homogeneous group of molecules. Generic binding of the Fc part of an antibody to protein A is often used for the first purification step in a purification process, in which a target protein is directly captured from clarified cell culture harvest media on a protein A affinity column. That single step removes most process-related impurities such as host-cell proteins (HCPs), DNA, and proteases, and the antibody can be eluted to more than 99% purity. High selectivity makes protein A affinity chromatography a perfect foundation of a MAb purification platform. Some existing purification processes comprise four chromatographic steps, but more commonly, three chromatography steps are used (3). The capture step using protein A is followed by different combinations of ionexchange chromatography, hydrophobicinteraction chromatography, and hydroxyapatite for intermediate purification and polishing. Here, we present part of a strategy to develop a platform for purification of MAbs (6) as well as an approach to optimizing that process. Our process uses three chromatography media selected from a toolbox of media for packed-bed chromatography. MabSelect and Capto media families for large-scale chromatography ( A TOOLBOX OF CHROMATOGRAPHY MEDIA The capture compartment of the toolbox contains the MabSelect family (MabSelect, MabSelect Xtra, and MabSelect SuRe). MabSelect contains recombinant protein A and has a dynamic binding capacity of about 30 mg/ml at a residence time of 2.4 minutes. MabSelect Xtra uses the same recombinant protein A ligand but has a smaller particle size and larger porosity, which results in an increased dynamic binding capacity. MabSelect SuRe is the youngest member of the MabSelect family. It contains an alkali-stabilized protein A derived ligand that can withstand harsh cleaning agents such as M NaOH. In addition to its alkaline stability, the ligand s higher protease stability compared with recombinant protein A further reduces ligand leakage (7, 8). A more generic elution for a broad range of MAbs and Fc fusion proteins has also been observed on MabSelect SuRe (9). All members of this family of media are based 48 BioProcess International JANUARY 2007

2 Figure 1: Monoclonal antibody purification platform on agarose matrices that display low nonspecific binding of host-cell proteins and result in a very pure antibody product. The base matrix also allows for high flow rates at low backpressures. In the reference process presented here, MabSelect SuRe was selected for capture because it allowed the medium to be cleaned with high concentrations of NaOH and provided homogenous elution conditions and low ligand leakage. In the intermediate polishing compartment of the toolbox are a number of chromatography media based on different chromatographic techniques. For the second step in the proposed platform process, we selected Capto S, a recently launched cationexchange chromatography medium. It is optimized for high capacities and flow rates. The S-ligand is coupled to the high-flow agarose base matrix using a dextran surface extender for increased capacity. The platform process concludes with Capto Q, an anion-exchange chromatography medium for polishing, which also was selected because of its high-flow and high-capacity properties (Figure 1). Our results show that MabSelect SuRe for capture followed by Capto S and Capto Q for removal and polishing will work effectively for the majority of antibodies and can be recommended as the first option when developing new antibody processes. The chromatography conditions described for each step can be used as a starting point when developing a purification process, but because all antibodies are different, some optimization will be required. Hydrophobic-interaction, thiophilic-interaction (10), or multimodal chromatography can be included in the process if removal of high levels of aggregates, generated upstream during Figure 2: A cycle on MabSelect SuRe including an intermediate wash step ( General Electric company, reproduced by permission) Sample: Murine myeloma (NS0) cell clarified feed containing 1.4 mg MAb/mL Loading Buffer: 20 mm Na-phosphate, 0.15 M NaCl, ph 7.4 Intermediate Wash: 25 mm Na-phosphate, 5% isopropanol, 0.5 M NaCl, ph 7.0 Elution: 20 mm Na-citrate, ph 3.7 Regeneration: 0.1 M Na-citrate, ph 3.0 Cleaning-in-Place: 0.5 M NaOH, 15 min. contact time Linear Flow Velocity: 250 cm/h, residence time 2.4 min. expression or in downstream purification, is required. CAPTURE WITH MABSELECT SURE The capture of antibodies using protein A affinity chromatography is a fairly straightforward unit operation. The basic principle is to bind them to the affinity medium at neutral or physiological ph and elute at an acidic ph. Even without optimization, an antibody can be eluted to high purity. Cell-culture optimization leads to increased antibody titers in the feeds. The current industry standard is 1 5 g/l (11). However, expression levels of at least 10 g/l are expected to be achieved in future commercial production (12). As antibody titers increase, a concomitant increase of impurities is expected. To meet this challenge, introduction of an intermediate wash step could be considered even when working with low nonspecific impuritybinding, agarose-based media such as MabSelect SuRe (Figure 3). Interactions between the impurities (e.g., host-cell proteins), the chromatography matrix (backbone of the medium and/or protein A ligand), and the antibody are likely to be a complex combination of intermolecular forces. Both hydrophobic and electrostatic interactions between impurities and the medium or the bound antibody can be expected. Because the strength of such interactions can be influenced by the composition of a buffer used in the wash step, that effect was investigated during development of the platform process. In this study, 96 different wash buffers were evaluated using a 96-well format. A broad range of buffer additives such as amino acids, detergents, and solvents in combination with different concentrations of NaCl at ph 5.0 and ph 7.0 were investigated. From the first 96-well screening, some buffer additives in combination with 0.5 M NaCl at ph 5.0 were identified as efficient in removing host-cell proteins from the MabSelect SuRe medium. However, the ph 5.0 buffers in combination with those additives and that salt concentration resulted in a decrease in recovery due to loss of MAb during the wash. Therefore, ph 7.0 buffers containing the same

3 Figure 5: Summary of fit for model of dynamic binding capacity at 10% breakthrough of target protein (QB10%) on Capto S ( General Electric company, reproduced by permission) Sample: 2.6 mg MAb Loading: 20 mm Na-phosphate, 0.15 M NaCl, ph 7.4 Elution: 20 mm Na-citrate, ph 5.0 to 3.0 for 20 CV Linear Flow Velocity: 150 cm/h JANUARY 2007 Figure 6: Coefficient plot for model with a 95% confidence interval ( General Electric company, reproduced by permission) BioProcess International Figure 7: Response contour plot for dynamic binding capacity at 10% breakthrough of target protein on Capto S ( General Electric company, reproduced by permission) An intermediate wash step can, in addition to increasing product purity, also extend the life of the MabSelect SuRe column. Furthermore, as a consequence of the increased product purity, the number of subsequent chromatography steps can possibly be reduced and thus enable a two-step chromatography process. Recently it has been shown that a more generic elution is obtained on MabSelect SuRe than with traditional protein A media (9). Figure 4 presents an example of the elution profiles of four different MAbs from MabSelect SuRe. The antibodies were eluted within a narrow ph range of The possibility of eluting the antibodies at a higher ph and in a narrower elution ph range supports the use of this medium in the proposed platform process. Figure 4: Elution ph for four different monoclonal antibodies on MabSelect SuRe ( General Electric company, reproduced by permission) additives and salt concentration were considered to be a better choice. The ph 7.0 buffers also gave a significant decrease of the host-cell protein levels in the eluates, but without reducing MAb recovery. As shown in Figure 3, the most promising wash buffers were further verified chromatographically. Different buffer additives such as arginine, isopropanol, polypropylene glycol, and Tween 20 in combination with 0.5 M NaCl at ph 7.0 gave a significant decrease in HCP levels in the eluates compared to a wash with 20 mm phosphate, 0.15 M NaCl, at ph 7.4, which was used as a control wash buffer. To further improve the results, design of experiments (DOE) could be considered for optimizing the wash step by exploring a broader ph and conductivity range in combination with a wider concentration range of some specific buffer additive (e.g., propylene glycol or propanol). 50 INTERMEDIATE P URIFICATION WITH CAPTO S In contrast with the capture and polishing steps in this platform process, the cation-exchange chromatography step requires more efforts in optimizing both binding and elution conditions. The Figure 3: Host-cell protein (HCP) levels in the MabSelect SuRe eluate pool after intermediate wash with various buffers at ph 7.0 ( General Electric company, reproduced by permission) Loading Buffer: 20 mm Na-nitrate, 116 mm NaCl, ph Sample: Pure CHO-derived MAb of the concentration 5 mg/ml Residence Time: 4 min.

4 The high selectivity of protein A chromatography used for capture enables a PLATFORM approach to purification of MAbs. binding is best optimized using DOE, which allows the effect of a number of different variables to be studied simultaneously. In our study, buffer ph, conductivity, and loading residence time were used as variables in optimizing binding conditions. DOE was performed using MODDE 7 software (Umetrics, www. umetrics.com). We applied a quadratic model (CCF), in which each selected experimental variable was varied within an interval, with one low value, one high value, and one center point. For example, the ph values of the experiments were studied in the interval ph , which meant that the low value was ph 4.5, the high value was ph 5.5, and the center point was ph 5.0. Conductivity was varied from 5 to 15 ms/cm and the residence time from 52 BioProcess International JANUARY 2007 two to six minutes. All three variables conductivity, ph in the loaded sample, and residence time turned out to have significant impact on the dynamic binding capacity. A model summary and a coefficient plot are shown in Figures 5 and 6, respectively. Figure 6 shows that conductivity and ph have stronger effects on the dynamic binding capacity than the residence time in the explored experimental space. From the model obtained in MODDE, Figure 9: Elution profiles of four different monoclonal antibodies from Capto ( General Electric company, reproduced by permission) Column: HiTrap Capto Q, 5 ml CV Sample: 500 µl MAb Loading: 20 mm Na-phosphate, 20 mm Tris, 20 mm glycin ph 11.0 Figure 7: Response contour plot for dynamic binding capacity at 10% breakthrough of target protein on Capto S ( General Electric company, reproduced by permission) Linear Flow Velocity: 100 cm/h Elution: 20 mm Na-phosphate, 20 mm Tris, 20 mm glycin gradient, ph 11.0 to 6.0 for 5 CV the dynamic binding capacity at 10% breakthrough (QB10%) was plotted as a function of loading ph and conductivity to further interpret the parameter influence. The result at four minutes is shown as a Figure 8: Capto S for intermediate purification: The start material, MabSelect SuRe purified monoclonal antibody, contained approximately 1,000 ppm host-cell proteins. The eluate from Capto S contained 40 ppm host-cell proteins at 98% yield ( General Electric company, reproduced by permission). Column: Sample: Load: Load and Wash: Elution: Capto S packed in a Tricom 5/200 column, bed height 20 cm (4 ml column volume) MabSelect SuRe purified MAb (NS0 derived) 90 mg/ml resin 20 mm Na-citrate, 8 mm NaCl, ph 4.8 (4 ms/cm) 20 mm Na-citrate, 66 mm MaCl, ph 5.3 (10 ms/cm) Strip: 20 mm Na-citrate, ph 5.3, 0.5 M NaCl Cleaning-in-Place: 1.0 M NaOH, contact time 15 min Linear Flow Velocity: Sample: Pure CHO-derived MAb of the concentration 5 mg/ml Loading Buffer: 20 mm Na-nitrate, 116 mm NaCl, ph Residence Time: 4 min. 500 cm/h, residence time 2.4 min throughout the method except during CIP (400 cm/h)

5 Figure 10: Flow-through profiles and yield for MAb #2 using different loading ph on Capto Q ( General Electric company, reproduced by permission) Column: Capto Q packed in a Tricom 5/100 column, bed height 10.5 cm (2.06 ml CV) Loading Buffer: 20 mm Na-phosphate, ph 7.0, 7.5, or 8.0 Sample: NS0-derived MAb, eluate from Capto S in 25 mm Na-phosphate, ph 7.5 Elution Buffer: 200 mm Na-phosphate, ph 8.0 Sample Load: 1 mg MAb/mL resin Linear Flow Velocity: 500 cm/h response contour plot in Figure 7. That plot shows that the highest dynamic binding capacity is obtained at ph at a conductivity of 5 6 ms/cm, where the capacity is 140 mg/ml. However, the window of operation is wide. Conductivities between 5 and 10 ms/cm and ph in the range of can be used to obtain capacities of at least 100 mg/ml. In most of the investigated design space, lower conductivity and ph values result in higher QB10% values. However, at ph 4.5, the capacity decreases as conductivity decreases below ~10 ms/ cm. This nontraditional behavior with low capacities at low conductivities can sometimes be even more prominent (data not shown), especially for large proteins on Capto S. The result presented in Figure 7 shows that if loading conditions are screened only at low conductivity, you can be misled to believe that the binding capacities on Capto S are low. Also, elution conditions can be optimized for the second step in the platform process. In Figure 8, we present results from such an optimization in which a 25-fold decrease in host-cell proteins was achieved by eluting the Figure 11: Capto Q for polishing operated in flow through mode. The flow-through fraction contained 5 ppm host-cell proteins, 0.7% aggregates, and Protein A (SuRe-ligand) below the level of quantification. The yield was 94%. ( General Electric company, reproduced by permission) Column: Capto Q packed in a Tricom 5/100 column, bed height 10 cm (1 ml CV) Sample: Capto S purified MAb (NS0 derived) Load: 125 mg/ml resin Load and Wash: 25 mm Na-phosphate, ph 7.5 (4.0 ms/cm) Regeneration: 25 mm Na-phosphate, 600 mm NaCl, ph 7.5 Cleaning-in-Place: 1.0 M NaOH, contact time 15 min. Linear Flow Velocity: 500 cm/h, residence time 1 min, 15 sec antibody with increased ph and conductivity in the elution buffer (Figure 8). POLISHING WITH CAPTO Q Most of the platform processes for MAbs include a Q-step because of its high efficiency in removing residual amounts of host-cell proteins, DNA, endotoxins, and viruses. Capto Q is a high-flow and high-capacity anion-exchange chromatography medium. Many of the biopharmaceutical MAbs are basic molecules with isoelectric points above 8. The nature of these molecules makes the polishing step with anion-exchange chromatography easy to operate in a flow-through mode. The basic antibody has a net positive charge below ph 8, whereas most of the host-cell proteins and other impurities such as DNA, endotoxins, and certain viruses are negatively charged and will consequently bind to a positively charged medium. However, it is not always easy to predict chromatographic behavior from knowledge of the isoelectric point (pi) of the protein. To better understand the binding elution conditions for a MAb on the anion-exchange chromatography medium, it is useful to perform a screening experiment in which the antibody is bound to the column and eluted with a linear ph gradient. Knowledge acquired from the elution profile can be used for determining nonbinding loading conditions on Capto Q. For optimal loading conditions, the highest possible loading ph should be used at which the antibody is still found in the flow-through and most of the JANUARY 2007 BioProcess International 53

6 impurities bind to the column. However, because of the risk of alkaline deamidation, a loading ph above 8.0 should be avoided. Most of the basic MAbs can be loaded at ph 7 8 at low conductivity. This loading condition will ensure binding of impurities and, at the same time, allow for a high recovery (>95%) of the antibody in the flow through. Figure 9 shows the elution profiles of four different antibodies with pi values ranging from 7.4 to 10, bound to Capto Q and eluted with a linear ph gradient. For the three most basic antibodies, a ph of 8.0 can be used in the polishing step. For MAb #2, with an isoelectric point of 7.4, it was necessary to screen several loading ph values to optimize for high recovery and efficient removal of impurities. Three different loading ph values were tested for this antibody: 7.0, 7.5, and 8.0 (Figure 10). At ph 8.0, an unacceptably high percentage of the antibody bound to the column, resulting in a very low step yield of 66%. At ph 7.0, the yield was quantitative, whereas at ph 7.5 (where the MAb was slightly interacting with the Capto Q medium) the yield was 96%. A ph of 7.5 was considered to be efficient for binding impurities while resulting in an acceptable yield and was therefore chosen as loading condition for MAb #2. Figure 11 shows results obtained from the optimized polishing step. The starting material was MAb #2 previously eluted from Capto S. Capto Q removed the residual amounts of host-cell proteins and leaked MabSelect SuRe ligand, resulting in acceptable purity levels for a subsequent formulation step. Although the load was 125 mg MAb per milliliter of medium, the upper load limit is expected to be much higher. It is possible to load about 400 mg MAb per milliliter of medium with no significant breakthrough of the host-cell proteins in flow-through fractions (data not shown). ACCELERATING DEVELOPMENT Establishing a purification platform comprising a standard set of unit operations and conditions will shorten process development times and increase the throughput of product candidates from research to clinic. The high selectivity of protein A chromatography used for capture enables a platform approach to 54 BioProcess International JANUARY 2007 Table 1: Summary of the process for MAb #2, an NS0-derived monoclonal antibody purification of MAbs. By including a postload wash before elution, the protein A step using MabSelect SuRe can be further improved. With the cationexchange chromatography media, it is possible to obtain capacities of above 100 g MAb per liter of medium in a wide conductivity and ph range that adds robustness to a process. Capto Q used in a flow-through mode for polishing removes the final impurities to levels acceptable for formulation. Table 1 summarizes the purification process for NS0-derived MAb #2. It presents the HCP, protein A, and aggregate concentrations as well as the yield after each chromatography step. After the final polishing step, the HCP concentration was 5 ppm, the concentration of soluble aggregates was 0.7%, and the protein A concentration was not quantifiable. The total yield after the three chromatography steps was 88%. MabSelect SuRe for capture followed by Capto S and Capto Q for removal and polishing can be recommended as the first option when developing new processes for purifying MAbs. ACKNOWLEDGMENTS Feeds and MAbs were kindly supplied by BioInvent International AB, Sweden; Polymun Scientific, Austria; and Boehringer Ingelheim, Germany. REFERENCES 1 Holmer AF. 324 Biotechnology Medicines in Testing Promise to Bolster the Arsenal Against Disease. PhRMA 2004 survey, 2 Walsh G. Biopharmaceutical Benchmarks Nat. Biotech. 24(7) 2006: Farid Suzanne S. Established Bioprocesses for Producing Antibodies As a Basis for Future Planning. Adv. Biochem. Engin./Biotechnol. Published online 8 June MabSelect SuRe Host-Cell Protein (ppm) 4 Pavlou AK, Belsey MJ. The Therapeutic SuRe-Ligand (ppm) Aggregate (%) Yield (%) Feed 750,000 NA NA NA 1, Capto S Not determined 98 Capto Q 5 Not quantifiable Antibodies Market to Eur. J. Pharm. Biopharm. 59(3) 2005: Slaff G. The Quest for Operational Excellence in Biopharmaceutical Manufacturing. Presented at the BioProcess International Conference, October 2004 (Boston, MA). IBC Life Sciences: Westborough, MA; 6 Grönberg A, et al. A Strategy for Development of a MAb Purification Platform. Poster presentation at the BioProcess International European Conference, February 2006 (Prague, Czech Republic). IBC Life Sciences: London, UK; 7 Johansson HJ. Advances in the Purification of Monoclonal Antibody Based Therapeutics. Presented at Antibody Development and Production, 1 3 March 2006 (Carlsbad, CA). IBC Life Sciences: Westborough, MA; 8 Jagschies G, et al. Technical and Economical Evaluation of Downstream Processing Options for Monoclonal Antibody (MAb) Production. Advances in Separation and Purification. BioPharm International (supplement to June 2006): Ghose S, et al. Antibody Variable Region Interactions with Protein A: Implications for the Development of Generic Purification Process. Biotech. and Bioeng. 92(6) 2005: Eriksson KO. Aggregate Removal from Monoclonal Antibodies By Thiophilic Aromatic Adsorption Chromatography. Presented at the ACS 232nd National Meeting and Exposition, September 2006 (San Francisco, CA). American Chemical Society: Washington, DC; 11 Ma N, et al. Development of a High Yield and Chemically Defined MAb Producing Process Through Medium and Feed Improvements. Presentated at the Waterside Conference 11th Annual Meeting, 1 3 May 2006 (Chicago, IL); 12 Birch JR, Racher AJ. Antibody Production. Engineered Antibody Therapeutics (Themed issue of Adv. Drug Delivery Rev.) 58(5 6) 2006: In press.

7 Corresponding author Anna Grönberg is a senior research engineer at GE Healthcare Bio-Sciences AB, Protein Separations R&D, GE Healthcare Bio-Sciences AB, Björkgatan 30, Uppsala, Sweden; , fax , anna.gronberg@ge.com; Elin Monié is a research engineer at GE; Maria Murby is now at Phadia AB ( Gustav Rodrigo is a scientist, Elisabeth Wallby is a research engineer, and Hans J. Johansson is a senior scientist, all at GE. MabSelect, MabSelect Xtra, MabSelect SuRe, Capto, UniFlux, Tricorn, and HiTrap are trademarks of GE Healthcare companies. Tween is a trademark of ICI Americas Inc. This article is based on the BEST POSTER chosen by editorial advisors at IBC s BioProcess International Conference and Exposition, February 2006, held in Prague, Czech Republic. BioProcess International invited the authors to adapt the poster for publication. Our thanks to all who participated! JANUARY 2007 BioProcess International 55