Abstract
The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the N-glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade®) was assessed and compared to two of its commercially available biosimilars (Remsima® and Inflectra®). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method.
Highlights
Recombinant monoclonal antibodies serve a fundamental role in the field of human therapeutics by providing highly efficacious therapies in crucial disease areas, such as oncology, auto-immune and skin diseases [1]
A good fit was observed between the values for several important N-glycan characteristics that can be considered as critical quality attribute (CQA)
The performance of middle-up hydrophilic interaction liquid chromatography (HILIC)-mass spectrometry (MS) analysis for the identification and quantification of the glycosylation pattern of therapeutic monoclonal antibodies (mAbs) was critically evaluated in comparison to two frequently practiced reference methods
Summary
Recombinant monoclonal antibodies (mAbs) serve a fundamental role in the field of human therapeutics by providing highly efficacious therapies in crucial disease areas, such as oncology, auto-immune and skin diseases [1]. Inherent to their manufacturing in cellular expression systems is the occurrence of numerous enzymatic and chemical posttranslational modifications (PTMs) [2]. Several distinct glycoforms, e.g., N-glycolylneuraminic acid or α1,3-bound galactose containing glycans, are associated with adverse immunogenic reactions [6,7] Taken together, this renders the glycan profile an important critical quality attribute (CQA) that requires comprehensive characterization to ensure safe and efficacious treatments for patients
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