Separation of mAbs molecular variants by analytical hydrophobic interaction chromatography HPLC: overview and applications

Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) is a powerful analytical method used for the separation of molecular variants of therapeutic proteins. The method has been employed for monitoring various post-translational modifications, including proteolytic fragments and domain misfolding in etanercept (Enbrel®); tryptophan oxidation, aspartic acid isomerization, the formation of cyclic imide, and α amidated carboxy terminus in recombinant therapeutic monoclonal antibodies; and carboxy terminal heterogeneity and serine fucosylation in Fc and Fab fragments. HIC-HPLC is also a powerful analytical technique for the analysis of antibody-drug conjugates. Most current analytical columns, methods, and applications are described, and critical method parameters and suitability for operation in regulated environment are discussed, in this review.

[1]  Bernhardt L Trout,et al.  Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ANS and Thioflavin T binding , 2011, mAbs.

[2]  G. Gilliland,et al.  Structure-based engineering of a monoclonal antibody for improved solubility. , 2010, Protein engineering, design & selection : PEDS.

[3]  Ronald T Borchardt,et al.  Aspartate isomerization in the complementarity-determining regions of two closely related monoclonal antibodies. , 2007, Biochemistry.

[4]  Dieter Söll,et al.  Natural expansion of the genetic code. , 2007, Nature chemical biology.

[5]  Bernhardt L. Trout,et al.  Design of therapeutic proteins with enhanced stability , 2009, Proceedings of the National Academy of Sciences.

[6]  D. Ejima,et al.  The effects of arginine on protein binding and elution in hydrophobic interaction and ion-exchange chromatography. , 2007, Protein expression and purification.

[7]  Liqiang Lisa Zhou,et al.  Impact of short range hydrophobic interactions and long range electrostatic forces on the aggregation kinetics of a monoclonal antibody and a dual-variable domain immunoglobulin at low and high concentrations. , 2011, International journal of pharmaceutics.

[8]  W. Xu,et al.  Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies , 2012, mAbs.

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

[10]  Alain Balland,et al.  Separation of populations of antibody variants by fine tuning of hydrophobic-interaction chromatography operating conditions. , 2008, Journal of chromatography. A.

[11]  B. Yan,et al.  O-fucosylation of an antibody light chain: characterization of a modification occurring on an IgG1 molecule. , 2008, Glycobiology.

[12]  Aditya A Wakankar,et al.  Analytical methods for physicochemical characterization of antibody drug conjugates , 2011, mAbs.

[13]  A. Alpert.  High-performance hydrophobic-interaction chromatography of proteins on a series of poly(alkyl aspart-amide)-silicas , 1986 .

[14]  P. Declerck.  Biosimilar monoclonal antibodies: a science-based regulatory challenge , 2013, Expert opinion on biological therapy.

[15]  D. Goldenberg,et al.  Microheterogeneity of a purified IgG1 due to asymmetric Fab glycosylation. , 1992, Molecular immunology.

[16]  Huub Schellekens,et al.  Structure-Immunogenicity Relationships of Therapeutic Proteins , 2004, Pharmaceutical Research.

[17]  K. Büssow,et al.  Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis , 2004, BMC Structural Biology.

[18]  J. Queiroz,et al.  Hydrophobic interaction chromatography of proteins. , 2001, Journal of biotechnology.

[19]  K. Inouye,et al.  Single-step purification of F(ab')2 mu fragments of mouse monoclonal antibodies (immunoglobulins M) by hydrophobic interaction high-performance liquid chromatography using TSKgel ether-5PW. , 1993, Journal of biochemical and biophysical methods.

[20]  Mingxiang Li,et al.  Separation of oxidized variants of a monoclonal antibody by anion-exchange. , 2011, Journal of chromatography. A.

[21]  Lei Zheng.  Monoclonal Antibodies: Development, Delivery and Applications , 2015 .

[22]  John F. Valliere-Douglass,et al.  Separation and characterization of an IgG2 antibody containing a cyclic imide in CDR1 of light chain by hydrophobic interaction chromatography and mass spectrometry. , 2008, Analytical chemistry.

[23]  K. Inouye,et al.  Single-step purification of F(ab')2 fragments of mouse monoclonal antibodies (immunoglobulins G1) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl-5PW. , 1992, Journal of biochemical and biophysical methods.

[24]  Marisa K Joubert,et al.  Classification and Characterization of Therapeutic Antibody Aggregates , 2011, The Journal of Biological Chemistry.

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

[26]  P. Tessier,et al.  Aggregation-resistant domain antibodies engineered with charged mutations near the edges of the complementarity-determining regions. , 2012, Protein engineering, design & selection : PEDS.

[27]  D. Ouellette,et al.  Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule , 2013, mAbs.

[28]  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.

[29]  Damon L. Meyer,et al.  Effects of Drug Loading on the Antitumor Activity of a Monoclonal Antibody Drug Conjugate , 2004, Clinical Cancer Research.

[30]  C A Smith,et al.  Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists. , 1993, Journal of immunology.

[31]  X. Geng,et al.  Liquid chromatography of recombinant proteins and protein drugs. , 2008, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[32]  John R. Engen,et al.  Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars , 2012, Nature Reviews Drug Discovery.

[33]  Å. Danielsson,et al.  An adaptation of hydrophobic interaction chromatography for estimation of protein solubility optima. , 1997, Journal of pharmaceutical and biomedical analysis.

[34]  Anthony Mire-Sluis,et al.  Characterizing biological products and assessing comparability following manufacturing changes , 2004, Nature Biotechnology.

[35]  W. Xu,et al.  Developability studies before initiation of process development , 2013, mAbs.

[36]  R J Harris,et al.  Heterogeneity of recombinant antibodies: linking structure to function. , 2005, Developments in biologicals.