Alternative mobile phase additives for the characterization of protein biopharmaceuticals in liquid chromatography – Mass spectrometry

When analyzing large complex protein biopharmaceuticals, ion-pairing agents imparting low pH are widely used as mobile phase additives to improve the chromatographic performance. However, one of the most effective additives in RPLC and HILIC, trifluoroacetic acid (TFA), is known as a strong suppressor of the MS signal and limits its use in hyphenated techniques. In this study, we evaluated a wide range of acidic additives to find alternatives to TFA that provided comparable chromatographic performance and improved MS sensitivity. It was observed that stronger acidic additives were required for intact level analysis compared to subunit level analysis and that the additive nature had a larger impact on the chromatographic performance in HILIC mode compared to RPLC. Therefore, four additives were identified as valuable alternatives to TFA in RPLC mode, namely, difluoroacetic acid (DFA), dichloroacetic acid (DClAA), trichloroacetic acid (TClAA), and methanesulfonic acid (MSA). Only one of these additives provided acceptable performance in HILIC mode, namely, TClAA.After evaluation of the MS performance, TClAA was discarded due to the apparent loss of intensity in both RPLC-MS and HILIC-MS mode. Together, these results demonstrate that for HILIC-MS analysis TFA remains the gold standard additive. However, DFA was found as promising alternative to TFA for RPLC-MS analysis and could play an important role in the development of methods for the characterization of the increasingly complex protein biopharmaceuticals

Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis

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

Isolation and identification of isocoumarin derivatives with specific inhibitory activity against Wnt pathway Lasiodiplodia venezuelensis and metabolome characterization of

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