Absolute Quantitation of Intact Recombinant Antibody Product Variants Using Mass Spectrometry

Abstract

Accurate and precise quantitative measurement of product-related variants of a therapeutic antibody is essential for product development and testing. Bispecific antibodies (bsAbs) are Abs composed of two different half antibody arms, each of which recognizes a distinct target, and recently they have attracted substantial therapeutic interest. Because of the increased complexity of its structure and its production process, as compared to a conventional monoclonal antibody, additional product-related variants, including covalent and noncovalent homodimers of half antibodies (hAbs), may be present in the bsAb product. Sufficient separation and reliable quantitation of these bsAb homodimers using liquid chromatography (LC) or capillary electrophoresis-based methods is challenging because these homodimer species and the bsAb often have similar physicochemical properties. Formation of noncovalent homodimers and heterodimers can also occur. In addition, since homodimers share common sequences with their corresponding halves and bsAb, it is not suitable to use peptides as surrogates for their quantitation. To tackle these analytical challenges, we developed a mass spectrometry-based quantitation method. Chip-based nanoflow LC-time-of-flight mass spectrometry coupled with a standard addition approach provided unbiased absolute quantitation of these drug-product-related variants. Two methods for the addition of known levels of standard (multi- or single-addition) were evaluated. Both methods demonstrated accurate and reproducible quantitation of homodimers at the 0.2% (w/w) level, with the single-addition method having the promise of higher analytical throughput.

A single mutation increases heavy-chain heterodimer assembly of bispecific antibodies by inducing structural disorder in one homodimer species

“…For comparison, KiH homodimers were prepared and assessed by SEC. Studies of KiH have reported covalent and noncovalent forms of KiH homodimers in which the noncovalent form lacks the disulfide bonds in the hinge but antibody halves are held together by interactions in the Fc (27,28). Noncovalent homodimers would be expected to migrate at the same V e as covalent homodimers by SEC but in nonreducing SDS-PAGE would disassemble into halves.…”

Recent advances in mass spectrometric analysis of glycoproteins

“…However, covalent and noncovalent knob homodimers in bsAb are undesired products, and it has been challenging to separate and quantify homodimers because these species and bsAbs often have similar physicochemical properties. An efficient strategy for unbiased quantitation of homodimers in bsAbs was introduced based on a standard addition approach using, knob hAbs (half antibodies) as a surrogate for the noncovalent knob homodimer, by chip‐based‐RPLC‐TOF‐MS analysis . The method demonstrated accurate and reproducible quantitation of homodimers resulting in precise quantitation of intact antibodies (Fig.…”

“…(A) Quantitation of intact recombinant antibody product variants using standard addition approach by MS. (B) Representative figures for MS data extraction, deconvolution, and normalization. Reprint with permission from .…”

Structural and Functional Characterization of a Hole–Hole Homodimer Variant in a “Knob-Into-Hole” Bispecific Antibody

“…Intact mass analysis has also been used for bispecific variants identification and quantitation. 9,11,12 Homodimers can potentially cause undesired bioactivity. In addition, because of their potentially unnatural structure and presumed low stability, the homodimers may be more prone to aggregate which may lead to increased immunogenicity.…”