Analytical protein a chromatography as a quantitative tool for the screening of methionine oxidation in monoclonal antibodies.

The presence of oxidized methionine residues in therapeutic monoclonal antibodies can potentially impact drug efficacy, safety, as well as antibody half-life in vivo. Therefore, methionine oxidation of antibodies is a strong focus during pharmaceutical development and a well-known degradation pathway. The monitoring of methionine oxidation is currently routinely performed by peptide mapping/liquid chromatography-mass spectrometry techniques, which are laborious and time consuming. We have established analytical protein A chromatography as a method of choice for fast and quantitative screening of total Fc methionine oxidation during formulation and process development. The principle of this method relies on the lower binding affinity of protein A for immunoglobulin G-Fc domains containing oxidized methionines, compared with nonoxidized Fc domains. Our data reveal that highly conserved Fc methionines situated close to the binding site to protein A can serve as marker for the oxidation of other surface-exposed methionine residues. In case of poor separation of oxidized species by protein A chromatography, analytical protein G chromatography is proposed as alternative. We demonstrate that analytical protein A chromatography, and alternatively protein G chromatography, is a valuable tool for the screening of methionine oxidation in therapeutic antibodies during formulation and process development.

[1]  Gang Huang,et al.  Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn , 2009, Protein science : a publication of the Protein Society.

[2]  Kent Axelsson,et al.  Oxidation of Human Insulin-like Growth Factor I in Formulation Studies: Kinetics of Methionine Oxidation in Aqueous Solution and in Solid State , 1996, Pharmaceutical Research.

[3]  Sanchayita Ghose,et al.  Antibody variable region interactions with Protein A: implications for the development of generic purification processes. , 2005, Biotechnology and bioengineering.

[4]  N. Brot,et al.  Biochemistry and physiological role of methionine sulfoxide residues in proteins. , 1983, Archives of biochemistry and biophysics.

[5]  M. Mølhøj,et al.  Simultaneous Assessment of Asp Isomerization and Asn Deamidation in Recombinant Antibodies by LC-MS following Incubation at Elevated Temperatures , 2012, PloS one.

[6]  Hongcheng Liu,et al.  Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody. , 2007, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[7]  Hongcheng Liu,et al.  Effect of methionine oxidation of a recombinant monoclonal antibody on the binding affinity to protein A and protein G. , 2008, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[8]  Kinetics of chemical degradation in monoclonal antibodies: relationship between rates at the molecular and peptide levels. , 2010, Analytical chemistry.

[9]  A. Pappenberger,et al.  Degradation of polysorbates 20 and 80: studies on thermal autoxidation and hydrolysis. , 2011, Journal of pharmaceutical sciences.

[10]  A. Hagman,et al.  Oxidation of Human Insulin-Like Growth Factor I in Formulation Studies, II. Effects of Oxygen, Visible Light, and Phosphate on Methionine Oxidation in Aqueous Solution and Evaluation of Possible Mechanisms , 1996, Pharmaceutical Research.

[11]  J L Cleland,et al.  Antioxidants for prevention of methionine oxidation in recombinant monoclonal antibody HER2. , 1997, Journal of pharmaceutical sciences.

[12]  P. Johns,et al.  Direct determination of methionine sulfoxide in milk proteins by enzyme hydrolysis/high-performance liquid chromatography. , 2007, Journal of chromatography. A.

[13]  C. Lowe,et al.  Design, synthesis, and application of a Protein A mimetic , 1998, Nature Biotechnology.

[14]  L. Björck,et al.  Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. , 1984, Journal of immunology.

[15]  Y. Shechter,et al.  Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide. , 1986, The Journal of biological chemistry.

[16]  E. Kabat,et al.  Sequences of proteins of immunological interest , 1991 .

[17]  B. Frangione,et al.  The amino acid sequence of a human immunoglobulin G3m(g) pFc' fragment. , 1977, Journal of immunology.

[18]  Ajit S. Narang,et al.  Reactive Impurities in Excipients: Profiling, Identification and Mitigation of Drug–Excipient Incompatibility , 2011, AAPS PharmSciTech.

[19]  E. Stadtman,et al.  Oxidation of methionine residues of proteins: biological consequences. , 2003, Antioxidants & redox signaling.

[20]  Andreas Petzold,et al.  Identification of Potential Sites for Tryptophan Oxidation in Recombinant Antibodies Using tert-Butylhydroperoxide and Quantitative LC-MS , 2011, PloS one.

[21]  J. Goding.  Use of staphylococcal protein A as an immunological reagent. , 1978, Journal of immunological methods.

[22]  Hanns-Christian Mahler,et al.  The Degradation of Polysorbates 20 and 80 and its Potential Impact on the Stability of Biotherapeutics , 2011, Pharmaceutical Research.

[23]  G. Kronvall,et al.  Definition of staphylococcal protein A reactivity for human immunoglobulin G fragments. , 1970, Immunochemistry.

[24]  J. Deisenhofer.  Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. , 1981, Biochemistry.

[25]  M. Ryadnov,et al.  FTIR markers of methionine oxidation for early detection of oxidized protein therapeutics , 2011, European Biophysics Journal.

[26]  Yang Wang,et al.  Impact of methionine oxidation in human IgG1 Fc on serum half-life of monoclonal antibodies. , 2011, Molecular immunology.

[27]  G J Kleywegt,et al.  Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG. , 1995, Structure.

[28]  C. Mohan,et al.  The Human IgG Subclasses , 2001 .

[29]  Y. Burstein,et al.  Selective oxidation of methionine residues in proteins. , 1975, Biochemistry.

[30]  C. Schöneich.  Methionine oxidation by reactive oxygen species: reaction mechanisms and relevance to Alzheimer's disease. , 2005, Biochimica et biophysica acta.

[31]  J. Sjöquist,et al.  Protein a from Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins , 1972, FEBS letters.

[32]  Hongcheng Liu,et al.  Structural effect of deglycosylation and methionine oxidation on a recombinant monoclonal antibody. , 2008, Molecular immunology.

[33]  B. Yan,et al.  HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region. , 2011, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.