Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth.
An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.
Battersby
J E
JE
Department of Analytical Chemistry, Genentech Inc., South San Francisco, CA 94080, USA. jeb@gene.com
Snedecor
B
B
Chen
C
C
Champion
K M
KM
Riddle
L
L
Vanderlaan
M
M
D000906:Antibodies / Q000032:analysis*; D002846:Chromatography, Affinity / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D005285:Fermentation; D007128:Immunoglobulin Fragments / Q000032:analysis*; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000032:analysis
D000906:Antibodies; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins