Development and scale‐up of the recovery and purification of a domain antibody Fc fusion protein‐comparison of a two and three‐step approach

A robust, economical process should leverage proven technology, yet be flexible enough to adopt emerging technologies which show significant benefit. Antibody and Fc‐fusion processes may capitalize on the high selectivity of an affinity capture step by reducing the total number of chromatographic steps to 2. Risk associated with this approach stems from the potentially increased time frame needed for process development as well as unforeseen changes in impurity profile during first scale‐up of drug substance (DS) for animal toxicology and clinical phase I trials (FIH) production, which could challenge a two‐step process to the point of failure. Two different purification strategies were pursued during process development for an FIH process of a dAB‐Fc fusion protein. A two‐step process was compared to a three‐step process. The two‐step process leveraged additives to maximize impurity reduction during affinity capture. While wash additives in combination with a mixed mode chromatography met all impurity reduction requirements, HCP levels were still higher as compared to the three‐step process. The three‐step process was implemented for manufacture of clinical material to mitigate risk. Biotechnol. Bioeng. 2015;112: 1417–1428. © 2015 Wiley Periodicals, Inc.

[1]  R. Carbonell,et al.  Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media. , 2011, Journal of chromatography. A.

[2]  Sanchayita Ghose,et al.  Evaluation and comparison of alternatives to Protein A chromatography Mimetic and hydrophobic charge induction chromatographic stationary phases. , 2006, Journal of chromatography. A.

[3]  Cheng Zhang,et al.  Caprylic acid precipitation method for impurity reduction: An alternative to conventional chromatography for monoclonal antibody purification , 2012, Biotechnology and bioengineering.

[4]  J. Lundblad,et al.  Inactivation of Lipid‐Enveloped Viruses in Proteins by Caprylate , 1991, Vox sanguinis.

[5]  Qi Chen,et al.  Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody production , 2010, Biotechnology progress.

[6]  R. Bayer,et al.  Recovery and purification process development for monoclonal antibody production , 2010, mAbs.

[7]  N. Sanaie,et al.  Applying high-throughput methods to develop a purification process for a highly glycosylated protein. , 2012, Biotechnology journal.

[8]  J. Warwicker,et al.  Evidence for the adaptation of protein pH-dependence to subcellular pH , 2009, BMC Biology.

[9]  E. Read,et al.  Viral clearance by flow‐through mode ion exchange columns and membrane adsorbers , 2014, Biotechnology progress.

[10]  René Gantier,et al.  Designing new monoclonal antibody purification processes using mixed-mode chromatography sorbents. , 2011, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[11]  V. Morais,et al.  A model mechanism for protein precipitation by caprylic acid: Application to plasma purification , 2012, Biotechnology and applied biochemistry.

[12]  Kurt Brorson,et al.  Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics , 2009, Biotechnology and bioengineering.

[13]  S. Lute,et al.  Analysis of viral clearance unit operations for monoclonal antibodies , 2010, Biotechnology and bioengineering.

[14]  G. Caraux,et al.  The modal distribution of protein isoelectric points reflects amino acid properties rather than sequence evolution , 2004, Proteomics.

[15]  Sanchayita Ghose,et al.  Comparison of the chromatographic properties of IgG1 and IgG2 antibody subclasses , 2010, Biotechnology and applied biochemistry.

[16]  J. Gutiérrez,et al.  Development and validation of a reverse phase HPLC method for the determination of caprylic acid in formulations of therapeutic immunoglobulins and its application to antivenom production. , 2009, Biologicals : journal of the International Association of Biological Standardization.

[17]  W. Nieuwenhuizen,et al.  Purification and properties of two lipases from pig adipose tissue. , 1976, Biochimica et biophysica acta.

[18]  J. Coffman,et al.  A tandem laboratory scale protein purification process using Protein A affinity and anion exchange chromatography operated in a weak partitioning mode , 2013, Biotechnology and bioengineering.

[19]  Bernhardt L Trout,et al.  Aggregation-prone motifs in human immunoglobulin G. , 2009, Journal of molecular biology.

[20]  Bernhardt L Trout,et al.  Developability index: a rapid in silico tool for the screening of antibody aggregation propensity. , 2012, Journal of pharmaceutical sciences.

[21]  R. Kyle,et al.  Two-dimensional gel electrophoresis of serum specimens from patients with monoclonal gammopathies. , 1982, Clinical chemistry.

[22]  A. Aruffo,et al.  CD40 and its ligand, an essential ligand-receptor pair for thymus-dependent B-cell activation. , 1992, Immunology today.

[23]  Lisa Connell-Crowley,et al.  Using high throughput screening to define virus clearance by chromatography resins , 2013, Biotechnology and bioengineering.

[24]  S. Ferrone,et al.  Re.: Purification of IgG monoclonal antibody by caprylic acid precipitation. , 1983, Journal of immunological methods.

[25]  G. Bishop,et al.  CD40 and autoimmunity: the dark side of a great activator. , 2009, Seminars in immunology.

[26]  Yuefeng Lu,et al.  Recent advancement in application of hydrophobic interaction chromatography for aggregate removal in industrial purification process. , 2009, Current pharmaceutical biotechnology.

[27]  M. Steinbuch,et al.  The isolation of IgG from mammalian sera with the aid of caprylic acid. , 1969, Archives of biochemistry and biophysics.

[28]  Bernhardt L Trout,et al.  Prediction of aggregation prone regions of therapeutic proteins. , 2010, The journal of physical chemistry. B.

[29]  K. Brorson,et al.  Identification of protein A media performance attributes that can be monitored as surrogates for retrovirus clearance during extended re-use. , 2003, Journal of chromatography. A.

[30]  P. Hinckley,et al.  Host cell protein clearance during protein a chromatography: Development of an improved column wash step , 2008, Biotechnology progress.

[31]  S. Ferrone,et al.  Purification of murine IgG monoclonal antibodies by precipitation with caprylic acid: comparison with other methods of purification. , 1989, Hybridoma.

[32]  A. Chanutin,et al.  The precipitation of plasma proteins by short-chain fatty acids. , 1960, Archives of biochemistry and biophysics.

[33]  P. Thomas,et al.  A simple, non-chromatographic purification procedure for monoclonal antibodies. Isolation of monoclonal antibodies against cytochrome P450 isozymes. , 1987, Journal of immunological methods.

[34]  E. Keshavarz‐Moore,et al.  Purification of antibodies using the synthetic affinity ligand absorbent MAbsorbent A2P , 2007, Nature Protocols.

[35]  J. Chandler.  Purification and Characterization of Antibodies , 2006 .

[36]  A. Chanutin,et al.  Albumin from heated human plasma. I. Preparation and electrophoretic properties. , 1954, Archives of biochemistry and biophysics.

[37]  Jie Chen,et al.  Comparison of standard and new generation hydrophobic interaction chromatography resins in the monoclonal antibody purification process. , 2008, Journal of chromatography. A.

[38]  Niccolò Taddei,et al.  Low-level expression of a folding-incompetent protein in Escherichia coli: search for the molecular determinants of protein aggregation in vivo. , 2010, Journal of molecular biology.

[39]  M. McKinney,et al.  A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. , 1987, Journal of immunological methods.

[40]  B. Poolman,et al.  The Role of Biomacromolecular Crowding, Ionic Strength, and Physicochemical Gradients in the Complexities of Life's Emergence , 2009, Microbiology and Molecular Biology Reviews.

[41]  Brian D. Kelley,et al.  High‐throughput screening of chromatographic separations: II. Hydrophobic interaction , 2008, Biotechnology and bioengineering.

[42]  Joe X. Zhou,et al.  Viral clearance using disposable systems in monoclonal antibody commercial downstream processing , 2008, Biotechnology and bioengineering.

[43]  H. Bull,et al.  Binding of fatty acids by proteins. , 1967, Archives of biochemistry and biophysics.

[44]  Jing Liu,et al.  A mechanistic study of Protein A chromatography resin lifetime. , 2009, Journal of chromatography. A.

[45]  F. Steindl,et al.  Comparison of protein A affinity sorbents III. Life time study. , 2006, Journal of chromatography. A.

[46]  Brian Kelley,et al.  Very Large Scale Monoclonal Antibody Purification: The Case for Conventional Unit Operations , 2007, Biotechnology progress.

[47]  Kurt Brorson,et al.  Bacteriophage and impurity carryover and total organic carbon release during extended protein A chromatography. , 2009, Journal of chromatography. A.