Isomerization in the CDR2 of a monoclonal antibody: Binding analysis and factors that influence the isomerization rate

Abstract

Isomerization of a monoclonal antibody is one of the common routes of protein degradation. An isomerization in the complementarity-determining region (CDR) was found previously and is investigated in depth in this work. Affinity analysis proves that the antibody with one isomerized heavy chain has lower binding. Binding constants were determined, and exhibited a slower on-rate in conjunction with a faster off-rate for this isomerization. To determine the role of the buffer on the rate of isomerization, this antibody was incubated in various matrices and the amount of isomerized antibody was determined by hydrophobic interaction chromatography (HIC). The rate was found to be dependent on the pH as well as the net negative charge of the buffer components that can act as proton acceptors. An Arrhenius plot was performed to predict the levels of isomerization and a comparison of real samples proved the model was correct. This work affirms that isomerization in the CDR of a therapeutic antibody is important to monitor and the formulation buffer plays a significant role in the rate of the isomerization.

Improving Trastuzumab’s Stability Profile by Removing the Two Degradation Hotspots

“…The deamidation hotspot at HC‐Asn328 under the acidic condition (pH 5.1 in this work) was not reported by Harris et al, which may be because of that trastuzumab was not stressed and tested in the acidic pH in their work. It is known that normally the deamidation of Asn residue proceeds favorably at neutral and basic pH values and the isomerization of Asp residue proceeds faster at acidic pH values (e.g., pH <6.0) . A special case of deamidation in acidic solution was recently reported for this Asn328 in VSNK peptide, in which deamidation was not detected in neutral pH .…”

“…Like other proteins, therapeutic mAbs are susceptible to a variety of chemical and physical degradation reactions during manufacturing, formulation storage and in vivo usage . Asparagines (Asn) deamidation and aspartate (Asp) isomerization are the most common pathways for the chemical degradation of mAbs, which could affect the in vitro stability, in vivo biological functions, and bioavailability of a therapeutic mAb . It has been reported that several IgG1 mAbs lost their bioactivities because of the deamidation and/or isomerization in their complementary determining regions (CDRs) .…”

“…[3][4][5][6][7][8][9] Asparagines (Asn) deamidation and aspartate (Asp) isomerization are the most common pathways for the chemical degradation of mAbs, which could affect the in vitro stability, in vivo biological functions, and bioavailability of a therapeutic mAb. [10][11][12][13][14] It has been reported that several IgG1 mAbs lost their bioactivities because of the deamidation and/or isomerization in their complementary determining regions (CDRs). [15][16][17][18][19][20][21] Cacia et al 11 reported that the Asp isomerization in the first light-chain (LC) CDR, namely, LC-CDR1, of an anti-IgE mAb resulted in approximately 80% loss in its binding affinity.…”

Protein asparagine deamidation prediction based on structures with machine learning methods

“…Such reaction makes Asp being considered as a protein hotspot of isomerization. Asp isomerization has been reported on antibodies by multiple papers [42–44]. Comparing to Asn deamidation, Asp isomerization has a higher rate at low pH (< 5.5) [1].…”

Application of Microchip Electrophoresis Sodium Dodecyl Sulfate for the Evaluation of Change of Degradation Species of Therapeutic Antibodies in Stability Testing

“…These heterogeneities include aggregation, fragmentation, deamidation, oxidation, N-and C-terminal differentiations. [2][3][4][5][6][7][8][9][10][11] Several analytical methods have been developed for these purposes, for example, SDS polyacrylamide gel electrophoresis for the determination of the purity of proteins, size exclusion chromatography for aggregation, ion exchange chromatography or hydrophobic interaction chromatography for the detection of deamidation and oxidation variants, and Ellman's assay for quantification of free thiol groups. 12,13 Capillary-based SDS electrophoresis (CE-SDS) is one version of the capillary gel electrophoresis (CGE) using soluble linear polymers.…”