Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.
Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.
Department of Chemical Engineering, McMaster University, Hamilton, ON, L8S 4L7, Canada.
Rahul
R
Sadavarte
Maureen
M
Spearman
Natalie
N
Okun
Michael
M
Butler
Raja
R
Ghosh
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000235:genetics / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies
EG2-hFc; chimeric heavy chain monoclonal antibody; glycan-analysis; hydrophobic interaction membrane chromatography; purification