Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion

Abstract

Monoclonal antibodies are subjected to a wide variety of post-translational modifications (PTMs) that cause structural heterogeneity. Characterization and control of these modifications or quality attributes are critical to ensure antibody quality and to define any potential effects on the ultimate safety and potency of antibody therapeutics. The biopharmaceutical industry currently uses numerous tools to analyze these quality attributes individually, which requires substantial time and resources. Here, we report a simple and ultrafast bottom-up liquid chromatography-mass spectrometry (uLC-MS) method with 5 min tryptic digestion to simultaneously analyze multiple modifications, including oxidation, deamidation, isomerization, glycation, glycosylation, and N-terminal pyro-glutamate formation, which can occur during antibody production in mammalian cell culture, during purification and/or on storage. Compared to commonly used preparation procedures, this uLC-MS method eliminates assay artifacts of falsely-increased Met oxidation, Asp isomerization, and Asn deamidation, a problem associated with long digestion times in conventional LC-MS methods. This simple, low artifact multi-attribute uLC-MS method can be used to quickly and accurately analyze samples at any stage of antibody drug development, in particular for clone and media selection during cell culture development.

A View on the Importance of “Multi-Attribute Method” for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy

“…Similarly, MAM can facilitate the monitoring of host cell proteins (HCPs) with immunogenicity or safety risks, and for any HCPs at unacceptable levels, MAM can aid the optimization of purification processes for greater clearance (32). In addition to being an excellent formulation screening tool, MAM is now widely used to monitor specific oxidation, deamidation, isomerization, and/or succinimide formation events that can impact product potency and establish appropriate product expiries to mitigate their impact (8,10,(12)(13)(14)32,33).…”

Development of a novel, high‐throughput screening tool for efficient perfusion‐based cell culture process development

“…The product quality was established using a multiattribute monitoring peptide mapping method. This method is similar to previous published examples . Protein samples (62 μg) are dried by centrifugal evaporation and then denatured with 75 μL solution of 6M Guanidine hydrochloride, 500 mM Tris, 1 mM EDTA at pH 8.2.…”

Maintaining consistent quality and clinical performance of biopharmaceuticals

“…While general improvements in analytical instrumentation and strategies have been achieved over the past few decades [14], the ability to detect and characterize variants and impurities in biopharmaceuticals has been facilitated to a large degree by advancements in mass spectrometry, with current instruments achieving high resolution detection of peptides in the femtomolar and attomolar range [43,44]. This technology is being leveraged to allow improved detection of functionally relevant glycans on therapeutic proteins [45][46][47], as well as the assessment of minor product variants potentially relevant to protein structure or function, such as sequence variants, truncations, incorrect disulfide bridge structures, and other amino acid modifications [48][49][50][51][52][53][54]. Further, mass spectrometry has become an orthogonal tool in the monitoring of process impurities such as host cell proteins (HCPs), providing improved assessment of the type and amount of these impurities and their potential impact on product stability and immunogenicity [55,56].…”