Antibodies are an important class of drugs, comprising more than half of all new FDA approvals. Therapeutic antibodies must be chemically stable both in storage and in vivo, following administration to patients. Therefore, to ensure patient safety, it is crucial to conduct impurity analysis and structural characterization of biopharmaceuticals for product reliability and consistency across the entire product life cycle. Quantitative analysis of various biologics modifications such as in antibodies for example glycosylation, deamidation, and oxidation is important in drug safety since these modifications could change the therapeutics bioactivity and potentially cause immunogenicity. Asparagine (Asn) deamidation is one of the main chemical modifications of interest for mAbs and is formed via a succinimide (Asu) intermediate that can be subsequently hydrolyzed to aspartic acid (Asp) or iso-aspartic acid (isoAsp, which is sensitive to environmental changes during the manufacturing process or long-term storage. In addition, deamidation of proteins such as many long-lived and disease-related proteins as a spontaneous non-enzymatic process in vivo has been widely studied in human degenerative diseases and natural aging. Rapid assessment strategies in early post-discovery processes to evaluate the chemical stability of therapeutic proteins are necessary to identify and correct particularly labile residues prior to committing time and resources towards accommodating such inherent risks downstream. LC/MS is commonly used for the relative quantitation of deamidation level based on the peak intensity or area ratio of the native and deamidated peptides. Absolute quantitation of deamidation is rarely investigated before and absolute quantitation of the succinimide intermediate has never been done before due to lack of standards.
Traditionally such testing, or protein quantitation, involves time-consuming preparation of synthetic isotope-labeled peptides which are used as internal standards to measure total protein concentrations in a sample helping researchers actively monitor the efficacy and safety of therapeutic protein components throughout the drug development process. To overcome this limitation, a coulometric mass spectrometry approach for absolute quantitation of proteins without the use of standards has been developed. The method instead applies liquid chromatography-mass spectrometry and an electrochemical flow cell to rapidly quantify and detect changes in target proteins or peptides based on electrochemical signatures.
In a new research paper published in the Journal Analytical Chemistry, New Jersey Institute of Technology researchers led by Professor Hao Chen unveiled a new method that can advance how pharmaceutical laboratories evaluate new protein-based drugs, such as therapeutic monoclonal antibodies being developed to treat a variety of diseases, from cancers to autoimmune diseases. Researchers say their electrochemistry-based approach could allow for safety and quality testing of up-and-coming biotherapeutics to be done at a fraction of the time required by conventional methods, which typically require the lengthy and costly production of certain biomaterials used for sample testing.
The research team explored coulometric mass spectrometry for multi-protein quantitation in one mixture sample in the same run and demonstrated the potential of coulometric mass spectrometry for absolute quantitation of low-abundant HCP in the presence of high-abundant NIST mAb 8671, without using standards. In addition, by using coulometric mass spectrometry. The study presents simultaneous quantitation of native and deamidated peptides as well as the succinimide intermediate from deamidation of Asn 318 residue of NIST 8671 mAb, for the first time.
The new method developed has the potential to have a major impact in quantitative proteomics, and it represents a paradigm shift in pharmaceutical industry in terms of monitoring biopharmaceutical product and process impurities for quality control. The authors demonstrated successfully their CMS method achieved absolute quantitation of multiple proteins (β- lactoglobulin B, α-lactalbumin and carbonic anhydrase) in a mixture in one run, without using any standards. Notably, the team also showcased the method’s capabilities for detecting protein deamidation a common degradation event in therapeutic proteins resulting from physical or chemical stresses throughout the manufacturing process and storage. Moreover, the team successfully quantified several protein degradation products, including a key intermediate of protein degradation the formation of succinimide which has never been done before with absolute quantification due to lack of standards, according to the study’s authors.
Future studies plan to apply their new method for largescale quantitation of thousands of proteins in one run. They also plan to improve the sensitivity of their CMS analysis to allow quantifying very low levels of proteins in complex biological samples, which could benefit research efforts ranging from clinical diagnostics and drug discovery to precision medicine for which identification and quantitation of samples at the molecular level is necessary.
Yongling Ai, Harsha P. Gunawardena, Xuanwen Li, Yong-Ick Kim, Howard D. Dewald, and Hao Chen. Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry. Anal. Chem. 2022, 94, 36, 12490–12499.