Impact of stirring material on formation of submicron and subvisible aggregates in mAbs by quantitative laser diffraction, dynamic light scattering and background membrane imaging.

Aggregation of monoclonal antibodies (mAbs) is the driving force for their undesirable immunogenic effects. There are multiple factors responsible for aggregation in therapeutic proteins. One significant cause is the process-related shear and interfacial stress generated due to impellers and stirrers. This investigation focuses on understanding the possible aggregation arising upon stirring mAb formulations using stirrers made of different materials. We used quantitative laser diffraction (qLD) to monitor and quantify the stirring induced formation of submicron and subvisible aggregates in the size range from 100 nm to 10 µm. We analysed various aspects of aggregate generation, such as onset of aggregation, particle size distribution, and concentration of aggregates generated using stirrers of different materials. We observed that mixing with stainless steel stirrers resulted in a quicker onset of aggregation and led to significantly higher concentrations of aggregates. Analysis of the stirred samples using dynamic light scattering (DLS) and background imaging technique (BMI) were conducted to complement the qLD analysis. All the three techniques resulted in a similar trend, showing presence of larger and higher quantities of aggregates in steel stirred samples, as compared to those stirred using PEEK and glass. Additionally, we performed SEC-HPLC to quantify the soluble fraction of monomer and recorded that the least amount was present in the steel stirred samples. This work highlights the need for optimizing the materials used for fabricating the stirrers/impellers.

Development and optimization of a LC-MS based multi-attribute method (MAM) workflow for characterization of therapeutic Fc-fusion protein.

The growing complexity of novel biopharmaceutical formats, such as Fc-fusion proteins, in increasingly competitive environment has highlighted the need of high-throughput analytical platforms. Multi-attribute method (MAM) is an emerging analytical technology that utilizes liquid chromatography coupled with mass spectrometry to monitor critical quality attributes (CQAs) in biopharmaceuticals. MAM is intended to supplement or replace the conventional chromatographic and electrophoretic approaches used for quality control and drug release purpose. In this investigation, we have developed an agile sample preparation approach for deploying MAM workflow for a complex VEGFR-targeted therapeutic Fc-fusion protein. Initially, a systematic time course evaluation of tryptic digestion step was performed to achieve maximum amino acid sequence coverage of >96.5%, in a short duration of 2 h, with minimum assay artifacts. This approach facilitated precise identification of five sites of N-glycosylation with successful monitoring of other CQAs such as deamidation, oxidation, etc. Subsequently, the developed MAM workflow with suitable tryptic digestion time was qualified according to the International council for harmonisation (i.e. ICH) Q2R1 guidelines for method validation. Post-validation, the analytical workflow was also evaluated for its capability to identify unknown moieties, termed as 'New Peak Detection' (i.e. NPD), and assess fold change between the reference and non-reference samples, in a representative investigation of pH stress study. The study, thus, demonstrated the suitability of the MAM workflow for characterization of heavily glycosylated Fc-fusion proteins. Moreover, its NPD feature could offer an all-encompassing view if applied for forced degradation and stability studies.

Mass spectrometry-based glycoprofiling of biopharmaceuticals by using an automated data processing tool: SimGlycan®.

The therapeutic and immunological properties of biopharmaceuticals are governed by the glycoforms contained in them. Thus, bioinformatics tools capable of performing comprehensive characterization of glycans are significantly important to the biopharma industry. The primary structural elucidation of glycans using mass spectrometry is tricky and tedious in terms of spectral interpretation. In this study, the biosimilars of a therapeutic monoclonal antibody and an Fc-fusion protein with moderate and heavy glycosylation, respectively, were employed as representative biopharmaceuticals for released glycan analysis using liquid chromatography-tandem mass spectrometry instead of conventional mass spectrometry-based analysis. SimGlycan® is a software with proven ability to process tandem MS data for released glycans could identify eight additional glycoforms in Fc-fusion protein biosimilar, which were not detected during mass spectrometry analysis of released glycans or glyco-peptide mapping of the same molecule. Thus, liquid chromatography-tandem mass spectrometry analysis of released glycans not only complements conventional liquid chromatography-mass spectrometry-based glycan profiling but can also identify additional glycan structures that may otherwise be omitted during conventional liquid chromatography-tandem mass spectrometry based analysis of mAbs. The mass spectrometry data processing tools, such as PMI Byos™, SimGlycan® , etc., can display pivotal analytical capabilities in automated liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry-based glycan analysis workflows, especially for high-throughput structural characterization of glycoforms in biopharmaceuticals.

Fast Gas Chromatography with Tandem Mass Spectrometry Analysis of Selected Persistent Organic Pollutants and Organophosphorus and Synthetic Pyrethroid Pesticides in Indian Prawn (Fenneropenaeus indicus) in Compliance with the EU-MRLs.

A fast GC with tandem MS method was developed and validated for multiresidue determination of 95 chemical contaminants (24 synthetic pyrethroids, 17 organochlorines, 17 organophosphorus compounds, 18 polycyclic aromatic hydrocarbons, and 19 polychlorinated biphenyls) in Indian prawns (Fenneropenaeus indicus) as per the European Union maximum residual limit requirements. Chromatographic separation and MS determination were achieved within a short run time of 18 min, without compromising sensitivity and specificity. Our findings revealed a 2.5× reduction in the run time compared with conventional GC methods. Sample preparation involved a QuEChERS-based extraction of 10 g sample with 10 mL acidified acetonitrile (1% acetic acid) and phase separation with 6 g anhydrous magnesium sulfate and 1.5 g sodium acetate. The extract was cleaned in two steps, first by dispersive cleanup with primary secondary amine and then by C18 SPE cartridge. The regression coefficients of linearity (r2) for the concentration range of 5-50 ng/mL were >0.99 for all the compounds. Recoveries at 5 and 10 ng/g levels were within the acceptable range of 70-120%. The repeatability (RSDr) and within-laboratory reproducibility (RSDwR) precisions were ≤20%. The method was successfully applied for analysis of the real world samples for incurred residues.