Purification strategies and platform alternatives for monoclonal antibodies

Transcription

1 Purification strategies and platform alternatives for monoclonal antibodies Annika Forss, Anna Grönberg, Anders Ljunglöf, Karin Torstenson GE Healthcare Life Sciences, Björkgatan 30, SE Uppsala, Sweden First presented at the BioProduction meeting, Dublin, Ireland October 22 23, 2013

2 Abstract High-titer feed-stocks and an increasing demand for separation of MAb variants (aggregates, fragments, charge variants, etc.) call for chromatography media (resins) with improved features. The focus here is to develop strategies for various Mab purification challenges. Options of different Protein A media and new high resolution polishing media, both ion-exchange and multi-modal, will be compared regarding binding/elution conditions and ability to separate MAbs from impurities. Depending on the level of impurities, different processes including one or two polishing steps will be proposed. 2

3 Introduction Platform approaches to downstream purification of MAbs have become generally established. Continuous improvement of these platforms with new chromatographic media with for example higher capacities and/or better selectivity constantly occur. Protein A chromatography media is commonly used as capture because of its robustness and high purity in the elution pool. The remaining impurities are being removed by either one or two polishing steps. In this work we present data from two purification platform processes, one 2-step and one 3-step process. Fig 1. Examples of 2 and 3 step purifi cation processes. 3

4 Capture step: Protein A media MabSelect SuRe or MabSelect SuRe LX are the first choices as capture due to the high purity, generic elution conditions and their alkali stability. The selection between MabSelect SuRe and MabSelect SuRe LX is mainly based on titer levels and productivity calculations. MabSelect SuRe LX is more suitable for high titer cell cultures with titers above 3 g/l and MabSelect SuRe more suitable for titer below 3 g/l. Fig 2. Cost performance comparison of protein A chromotography media. Courtesy: Shohei Kobayashi, Chugai, Japan. 4

5 2-step process: Polishing with Capto adhere ImpRes (FT) The optimal flow through (FT) conditions for MAb1 were identified from screening in 96 well filter PreDictor plates. Further optimization was performed in small scale columns using Design of Experiments. ph was varied between 4.7 and 5.7, ionic strength (IS) between and 0.42 M and load between 60 and 100 g/l. The responses were aggregate, HCP and yield. Data was evaluated with MODDE software. The contour plot show low aggregate content at high ph and high IS, low HCP content at high ph and low IS and highest yield at low ph and low IS. 5

6 2-step process: Polishing with Capto adhere ImpRes (FT) The green area in the sweet spot analysis represents the conditions where all three criteria are met. 6

7 2-step process: Polishing with Capto adhere ImpRes (FT) The predicted run corresponds well to the verification run and all three target criteria were met (Table 1). 7

8 3-step process: Polishing with Capto SP ImpRes Capto Q Screening of binding conditions for MAb2 was performed in 96 well format. The varied conditions were ph and NaCl concentration (Fig 5). Red area corresponds to the highest static binding capacities and blue the lowest capacities. ph 5 and 5.5 were chosen as binding conditions. Elution strategies (represented by white arrows) were selected and tested in small scale columns. The comparison between elution at ph 5 an ph 5.5 showed similar yield but better aggregate clearance at ph 5. Based on the gradient elution experiment a step elution protocol was developed for Capto SP ImpRes at ph 5 (Fig 6). Fig 5. Contour plot from screening in Predictor Capto SP ImpRes 6 μl. 8

9 3-step process: Polishing with Capto SP ImpRes Capto Q The results showed that aggregates and HCP were efficiently removed with high MAb yield. Capto Q was added to the process to further reduce the HCP levels. Yield and purity are presented in Table 2. 9

10 Discussion Both the 2- and 3-step process presented here, efficiently removed aggregates and HCP at a high yield. If there is a need for increased resolution in the 2-step process Capto adhere ImpRes can be used in B/E elute mode instead of FT, as presented earlier in Application Note An alternative (Fig 7) to Capto SP ImpRes is the multimodal cation exchanger Capto MMC ImpRes. Capto MMC ImpRes has different selectivity compared to Capto SP ImpRes. It also has a wider window of operation in terms of ph and conductivity. This could be useful for example for MAbs not stable at loading conditions for CIEC. This might be especially important for molecules that are sensitive to acidic ph s. For additional impurity/aggregate clearance, Capto adhere or Capto adhere ImpRes can be used as an alternative to Capto Q in a 3-step process, especially when the aggregate clearance is not sufficient after the two first purification steps. 10

11 Platform Alternatives 2-step process 3-step process MabSelect SuRe MabSelect SuRe LX MabSelect SuRe MabSelect SuRe LX Capto adhere ImpRes (B/E) Capto MMC ImpRes Capto adhere (FT) Capto adhere ImpRes (FT) Capto adhere ImpRes (B/E) Fig 7. Toolbox alternatives for challenging purifications. 11

12 Conclusion Based on the results of the selection of alkali-stabilized Protein A media for capture and the development of polishing step/steps for two different monoclonal antibodies, the following conclusions can be made: The selection between MabSelect SuRe and MabSelect SuRe LX should be based on titer levels and productivity calculations. Capto adhere ImpRes in flow trough mode efficiently removed MAb aggregates and HCP in a two step purification process for MAb1. The combination of Capto SP ImpRes and Capto Q for polishing reduce the aggregates and HCP content to acceptable levels in an optimized purification process for MAb2. Platform alternatives could be used for challenging purifications (Fig 7). 12

13 Acknowledgments GE, imagination at work, and GE monogram are trademarks of General Electric Company. Capto, MabSelect SuRe, and PreDictor are trademarks of GE Healthcare companies. Modde is a trademark of Umetrics AB. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information General Electric Company All rights reserved. First published October 2013 GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden 13