Kemwell’s Expertise in Size Exclusion Chromatography for Isoform Separation of a Novel Tri-specific Antibody

Introduction:

Size variants determination is a critical quality attribute for a therapeutic recombinant antibody, since they can impact the drug product safety, potency, and efficacy. Size exclusion chromatography is the most frequently employed technique for estimation of size variant and SE-HPLC principally separates out variants based on size, shape and molecular weight. A novel tri-specific antibody is recombinantly produced in Chinese hamster ovary (CHO) cells. Our studies exemplify the effectiveness of native SE-HPLC for separation of isoform variant (*P1) of Novel Tris-specific antibody. The study involves development of SE-HPLC method for Tri-specific antibody to differentiate the HMWP and the isoform variant of Tri-specific antibody. SEC-MALS and functional assay by ELISA results demonstrated that *P1 peak observed in SEHPLC contains structural isoform variant with molecular weight similar to main peak, and the enriched *P1 peak fraction had an equal potency to the main peak, confirming this isoform variant has similar access to the antigen-binding site.

Objective:

To identify and demonstrate the identity of *P1 peak observed in SE-HPLC. To demonstrate the potency of *P1 peak in comparison to main peak

Approach:

Size variants determination is a critical quality attribute of a therapeutic recombinant antibody, since the size variant can impact the drug product safety, potency, and efficacy. Size exclusion chromatography is most frequently employed for estimation of size variant and SEC principally separates out variants based on size, shape and molecular weight.

A Novel Tri specific antibody is recombinantly produced in Chinese hamster ovary (CHO) cells and based on SEC principle; our studies exemplify the effectiveness of native SEC for separation of hydrodynamic variant/isoform variant (Peak A) of Novel Trispecific antibody. The study involves development of SEC method for Trispecific antibody to differentiate the HMWP form of Tri specific antibody and the isoform form of Trispecific antibody. Electrospray ionization-mass spectrometry (ESI-MS), Capillary electrophoresis, Fluorescence and SEC-MALS results demonstrated that SE-HPLC Peak A contains structural isoform variant. Isolated Peak A fraction had an equal potency to the Main peak, confirming this isoform variant has similar access to the antigen-binding site

Conclusion:

1. The enriched Main peak and enriched *P1 + P1 + P2 were shown to have biological activity comparable to that of the control. On the other hand, there is no biological activity or potency in Enriched P3 and P4.
2. Confirmed by the SEC-MALS result, the peak left to Main peak peak (*P1) is not an HMW impurity peak and identified as main peak's conformational isoforms. The differential elution of *P1 isoforms could be attributed to differential size/shape in comparison to main peak
3. Further confirmation on isoforms will be carried out by HOS techniques like, NMR and HDX-MS