Optimizing Antibody Enrichment for Pharmacokinetic Assays

Schematic showing immuno-enrichment using High Capacity Magne® Streptavidin Beads.

Schematic showing immuno-enrichment using High Capacity Magne® Streptavidin Beads.

During preclinical research and development of therapeutic antibodies, multiple variants of each antibody are assessed for pharmacokinetic (PK) characteristics across model systems such as rodents, beagles and primates. Ligand-binding assays (LBA) or liquid chromatography coupled to tandem mass spectrometry(LC–MS/MS)-based methods represent the two most common technologies used to perform the PK studies for mAb candidates(1,2).

Using either method it is essential to ensure accurate quantitative results that the initial enrichment of the target therapeutic antibody from serum or plasma be optimal. Biotinylated antibodies or antigens (against the therapeutic targets) immobilized onto high capacity streptavidin beads will enrich therapeutic antibody from serum or plasma samples. (Figure13666MC.eps). The affinity of biotin for streptavidin (Kd = 10–15) is one of the strongest and most stable interactions in biology therefore the biotin-streptavidin interaction cannot be reversed under non-denaturing conditions. Hence, it is possible to perform extensive washing to remove nonspecifically bound protein and elute therapeutic antibodies without also eluting the biotinylated component, thus improving the detection limit.

Magnetic based separation techniques have several advantages in comparison with standard separation procedures. This process is usually very simple, with only a few handling steps. All the steps of the purification procedure can take place in one single test tube. The magnetic separation techniques are also the basis of various automated procedures. Learn more about  the High Capacity Magne™ Streptavidin Beads (Cat # V7820) .


Purification of biotinylated proteins

Biotinylation is an attractive approach for protein complex purification due to the very high affinity (Kd = 10–15 M) of avidin/streptavidin for biotinylated templates. With typical avidin or streptavidin, the biotin-binding affinity is so great that purification with these traditional media require denaturing conditions for elution,such as 8 M Guanidine•HCl at pH 1.5 or boiling in reducing SDS-PAGE sample loading buffer. To avoid these harsh conditions SoftLink™ Soft Release Avidin resin can be used. These particles consist of monomeric avidin coupled to a methylacrylate resin.

This resin provides the same specificity of binding to biotin afforded by tetrameric biotin, but enables the release of biotinlylated molecules under mild nondenaturing conditions (5mM biotin).

The following are recent references that used the SoftLink™ Resin for the noted application:

Kashwayama, Y. et al. (2010) Identification of a substrate-binding site in a peroxisomal beta-oxidation enzyme by photoaffinity labeling with a novel palmitoyl derivative. J. Biol. Chem. 285, 26315–25. (Purification of photoaffinity labeled proteins for subquenant binding/activity experiments)

Takahashi, M. et al. (2010) Tailor-made RNAi knockdown against triplet repeat disease-causing alleles. Proc. Natl. Acad. Sci. 107, 21731-36 (Innovated procedure using biotin labeled cDNAs for the identification of nucleotide variations)

Kress, D. et al. (2009) An asymmetric model for Na+-translocating glutaconyl-CoA decarboxylases. J. Biol.Chem. 284, 28401–9 (Purification of Clostridium biotin carrier proteins that play a role in decarboxylation)

Akahori, Y. et al. (2009) Characterization of neutralizing epitopes of varicella-zoster virus glycoprotein H. J. Virol. 83, 2020–4. (purification of double stranded cDNA fragments amplified by PCR with a biotin-tagged PCR primer)

Shonsey, E.M. et al. (2008) Inactivation of human liver bile acid CoA:amino acid N-acyltransferase by the electrophilic lipid, 4-hydroxynonenal. J. Lipid Res. 49, 282–94 (purification of recombinant protein expressed in E.coli containing C-terminal avidin tag)

Andachi, Y. et al. (2008) A novel biochemical method to identify target genes of individual microRNAs: identification of a new Caenorhabditis elegans let-7 target. RNA 14, 2440–51 (purification of double stranded cDNA fragments amplified by PCR with a biotin-tagged PCR primer)

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