Rapid Purification of Human Bispecific Antibodies via Selective Modulation of Protein A Binding

Autor: Dennis R. Goulet, Rose Decker, Jose Pardinas, Susan H. Tam, Eva Emmell, Mark L. Chiu, Songmao Zheng, Mehabaw G. Derebe, Catherine N. Leettola, Adam Zwolak
Jazyk: angličtina
Rok vydání: 2017
Předmět:
0301 basic medicine
Models
Molecular
medicine.drug_class
Mutant
Gene Expression
lcsh:Medicine
Plasma protein binding
medicine.disease_cause
Monoclonal antibody
Protein Engineering
Chromatography
Affinity
Protein Structure
Secondary
Article
03 medical and health sciences
Mice
Antibodies
Bispecific
medicine
Animals
Humans
Protein Interaction Domains and Motifs
Amino Acid Sequence
Binding site
Staphylococcal Protein A
lcsh:Science
Mutation
Multidisciplinary
Binding Sites
biology
Chemistry
Protein Stability
Receptors
IgG
lcsh:R
Hydrogen-Ion Concentration
Fragment crystallizable region
Molecular biology
In vitro
Recombinant Proteins
Immunoglobulin Fc Fragments
Kinetics
030104 developmental biology
HEK293 Cells
Amino Acid Substitution
Immunoglobulin G
biology.protein
lcsh:Q
Protein A
Half-Life
Protein Binding
Zdroj: Scientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
Scientific Reports
ISSN: 2045-2322
DOI: 10.1038/s41598-017-15748-0
Popis: Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or “TLQ”) in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro, and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo studies.
Databáze: OpenAIRE
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje