Identification and characterization of a residual host cell protein hexosaminidase B associated with N‐glycan degradation during the stability study of a therapeutic recombinant monoclonal antibody product

Abstract

Host cell proteins (HCPs) are process-related impurities derived from host organisms, which need to be controlled to ensure adequate product quality and safety. In this study, product quality attributes were tracked for several monoclonal antibodies (mAbs) under the intended storage and accelerated stability conditions. One product quality attribute not expected to be stability indicating is the N-glycan heterogeneity profile. However, significant N-glycan degradation was observed for one mAb under accelerated and stressed stability conditions. The root cause for this instability was attributed to hexosaminidase B (HEXB), an enzyme known to remove terminal N-acetylglucosamine (GlcNAc). HEXB was identified by liquid chromatography-mass spectrometry (LC-MS)-based proteomics approach to be enriched in the impacted stability batches from mAb-1. Subsequently, enzymatic and targeted multiple reaction monitoring (MRM) MS assays were developed to support process and product characterization. A potential interaction between HEXB and mAb-1 was initially observed from the analysis of process intermediates by proteomics among several mAbs and later supported by computational modeling. An improved bioprocess was developed to significantly reduce HEXB levels in the final drug substance.A risk assessment was conducted by evaluating the in silico immunogenicity risk and the impact on product quality. To the best of our knowledge, HEXB is the first residual HCP reported to have impact on the glycan profile of a formulated drug product. The combination of different analytical tools, mass spectrometry, and computational modeling provides a general strategy on how to study residual HCP for biotherapeutics development.

Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling

“…HCPs can copurify with the therapeutic protein via specific or nonspecific interactions. 10,11 Several lipases, such as PLBL2, LPL, and LPLA2, have been reported to degrade PS-80 or PS-20 in biotherapeutics. [4][5][6][7]9 Multiple analytical tools have been developed as part of an overall HCP control strategy.…”

“…PS prevents agitation-induced aggregation, and minimizes surface adsorption. − Polysorbate-80 (PS-80) and polysorbate-20 (PS-20) are the two most widely used PSs in the biopharma industry. , The degradation of PS can result in turbidity and formation of subvisible particles, which impact product quality or shorten the shelf life of the drug product. , PS degradation is an industry-wide challenge for process and formulation development in biotherapeutics. − The challenge increases with higher cell density and titer in bioprocess as well as higher drug concentrations in formulation development. , There are multiple mechanisms for PS degradation which can be grouped into two main categories: oxidation and hydrolysis. , Enzymatic hydrolysis is the dominant mechanism for PS degradation, which results in cleavage of an ester bond in PS by lipases, esterases, or possibly other host cell proteins (HCPs) without known lipase activity. HCPs can copurify with the therapeutic protein via specific or nonspecific interactions. , Several lipases, such as PLBL2, LPL, and LPLA2, have been reported to degrade PS-80 or PS-20 in biotherapeutics. − , …”

“…ELISA provides one summed value for total HCP content. In some cases, specific antibodies were developed to known HCPs present in a sample. , Alternatively, liquid chromatography–mass spectrometry (LC-MS) based proteomics approaches are evolving rapidly as an orthogonal assay for HCP characterization, since proteomics can identify and quantify practically any individual HCP with certain abundance levels. ,− Absolute quantification of individual HCPs can be achieved by multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) targeted approaches with suitable internal standards. , However, current proteomics approaches have two main analytical challenges to overcome when investigating the cause of PS-80 degradation.…”

The measurement and control of high-risk host cell proteins for polysorbate degradation in biologics formulation

“…The identification of individual HCPs is critical for Quality by Design-based process development (supports development of a control strategy) and risk assessment. Traditional abundance-based proteomics (TABP) has been demonstrated as a valuable tool to identify and track the clearance of PSDEs and other HCPs throughout a purification process, which supports process development efforts (both upstream and downstream) on key HCPs that can impact product quality and stability [ 5 , 24 ].…”

Annotation of the non-canonical translatome reveals that CHO cell microproteins are a new class of mAb drug product impurity

“…Host cell proteins (HCPs) present in the final drug product are a particular concern, due to the risk that a HCP could elicit an immune response in the patient or reduce efficacy (Hanania et al, 2015). In addition, the presence of proteolytic HCPs can degrade or affect the stability of the mAb (Li et al, 2021; Luo et al, 2019). Regulatory authorities consider the amount of HCP in the final product to be a critical quality attribute, and require that the total HCP concentration be < 100 ppm (Bracewell et al, 2015).…”