Sensitive methods for evaluation of antibodies for host cell protein analysis and screening of impurities in a vaccine process Christine Sund Lundströma, Anna Mattssona, Karin Lövgrenb, Åsa Sundberga, Tryggve Bergandera, Anneli Jorsbacka, Karin Söderquista, Maria Winkvista, Mats Lundgrena, Camilla Estmer Nilssona aGE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden bIsconova, Kungsgatan 109, SE-753 18, Uppsala, Sweden First presented at the Vaccine Technology IV conference in Albufeira, Portugal May 20-25, 2012

Introduction In order to provide a reliable host cell protein (HCP) estimation, a mixture of polyclonal antibodies with broad affinity is beneficial, e g covering a large part of the host cell proteome. Here we show an evaluation of the affinity spectra of HCP antibodies purified from rabbits immunized with Vero cell lysate using two different adjuvants: FCA and AbISCO®. The specific antibodies were first purified on Vero cell lysate-coupled columns and then coupled as ligands on columns to capture the specific Vero cell proteins targeted (inverse Western, figure 1). The purified proteins were CyDye™ labeled and analyzed by electrophoresis. The specific antibodies were also analyzed with standard Western blotting and compared with the inverse Western. A commercial mixture of purified specific antibodies for Vero cell lysate was used as control.

Introduction The specific antibodies were evaluated in Biacore™ T200 as a HCP concentration analysis, using Vero cell lysate as reference (figure 2). Samples from influenza virus purification process were analyzed with the Biacore HCP assay and with a commercial ELISA for Vero cell proteins. Figure 3 shows screening of host cell derived impurities throughout an influenza vaccine process using 2-D DIGE analysis, analyzing protein pattern obtained for different routes in process development.

Schematic Principle TM

Anti-HCP pAB Evaluation Figure 1. Standard (WB) and inverse Western (IW) blot on purified HCP specific antibodies. (A) WB. Vero cell lysate was run on SDS-PAGE, blotted to nitrocellulose filter and detected with (I) purified HCP specific antibodies (FCA and AbISCO) and (II) Cy-labelled anti-rabbit antibodies. (B) IW. Purified HCP specific antibodies (FCA, AbISCO, Commercial) were coupled to NHS-activated Sepharose™ HP and Vero cell lysate specific proteins were purified, Cy-Dye™ labelled and separated on SDS-PAGE. All gels were scanned for CyDye in Typhoon™ FLA 9000 . (C) Vero cell lysate were run on SDS-PAGE and post stained with GelCode Blue (Pierce). pAb= polyclonal antibody.

Biacore™ HCP Assay Figure 2. Assay principle: (A) Anti-HCP antibodies (green) were immobilized on a sensor chip. Sample was injected and specific HCP bound to the immobilized antibodies, resulting in a response. (B) Overlay plot showing sensor-grams of injected HCP standard. Response levels were measured between report points (marked as X) before and after injection. After each injection the surface was regenerated in preparation for a new injection. Standard curve from Vero HCP analysis: (C) Report points were plotted versus the concentration. (D) Calibration trends in T200 software. Each sample value was calculated after the calibration trend at the specific cycle.

Biacore™ HCP Assay Standard1 HCP (µg/ml) Biacore HCP CV (%, n=6) Table 1. Recovery of standard in Biacore HCP assay. Table 2. HCP analysis of samples from influenza virus process. Standard1 HCP (µg/ml) Biacore HCP CV (%, n=6) Recovery (%) 40 2.0 100 20 1.6 101 10 1.0 99 5 0.8 2.5 0.6 1.25 1.3 0.4 0.625 0.62 0.313 0.30 4.2 96 Sample Biacore HCP (µg/ml) ELISA2 Bradford Total protein Control3 86 91 225 Harvest 39 na 79 UF/DF 80 143 132 Flowthrough 8.0 8.2 <LOD 2) Vero HCP ELISA from Cygnus Technologies 3) A UF/DF sample from A/H1N1/Solomon Islands processing. Used as control in several analyses. na = not analyzed, LOD= Limit of detection Table 3. Comparison of analysis time. Analysis time 10 samples, 3 dilutions, 2 replicates, standards Biacore HCP ELISA2 Total 16h 6h Hands on 1h 4h 1) Vero cell lysate

2-D DIGE OF HCP IN INFLUENZA VIRUS PROCESS

Conclusions The selection of adjuvant will affect the antibody target profile. HCP proteins detected in purified virus: virus proteins or ”impurities”? [Shaw, ML. et al. Cellular proteins in influenza virus particles. 2008. PLOS Pathogens. Vol. 4, Issue 6.]

Conclusions Biacore™: Specific protein quantification confidence in mass balance calculations Short hands-on time In real time analysis; partial results in <10min WB vs IW: IW gives several analyses possibilities of pAb target profile, as proteins are purified With IW several antibodies can be compared in the same electrophoresis gel CyDye™ multiplexing simplify protein pattern comparisons 2-D DIGE: High sensitivity and broad dynamic range. Less hands-on; no need of post staining or western blotting. Detection is independent of antibody performance. Multiplexing with different CyDyes direct study of impurity profile.

Acknowledgments GE, imagination at work, and GE monogram are trademarks of General Electric Company. HiTrap, Sepharose, Biacore, CyDye, Image Quant, Typhoon and DeCyder are trademarks of GE Healthcare companies. All third party trademarks are property of their respective owners. © 2012 General Electric Company―All rights reserved. First published 2012. All goods and services are sold subjects 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. GE Healthcare Bio-Sciences AB, Björkgatan 30, 751 84 Uppsala, Sweden. www.gelifesciences.com/bioprocess