Exposing the molecular heterogeneity of glycosylated biotherapeutics.

The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.

Challenges with development of a pharmacokinetics assay to measure a variably glycosylated fusion protein.

Aim: Development of recombinant fusion proteins as drugs poses unique challenges for bioanalysis. This paper describes a case study of a glycosylated fusion protein, where variable glycosylation, matrix interference and high sensitivity needs posed unique challenges. Results: Six different assay configurations, across four different platforms were evaluated for measurement of drug concentrations. Two platforms that achieved the assay requirements were Simoa HD-1 and immune-capture LC-MS/MS-based assay. Conclusion: Both, Simoa HD-1 and the mass spectrometry-based methods were able to detect total drug by providing the adequate matrix tolerance, required sensitivity and detection of all the various glycosylated fusion proteins to support clinical sample analysis. The mass spectrometry-based method was selected due to robustness and ease of method transfer.

DNA spike studies for demonstrating improved clearance on chromatographic media.

DNA spike clearance methods were used to demonstrate improved clearance factors on anion exchange and hydrophobic interaction columns used in the production of human therapeutic proteins. DNA clearance at large-scale was first measured for a monoclonal antibody expressed in Chinese Hamster Ovary (CHO) cells and an antibody fragment expressed in Escherichia coli. Small-scale spike experiments were then performed on individual chromatographic steps using host-specific DNA paired with TaqMan PCR assay methods. This approach has advantages of improved specificity, sensitivity, cost and throughput compared to other types of spike clearance methods. The anion exchange column used in the monoclonal antibody process was shown to have very high capacity for CHO DNA, resulting in greater than 7.1 log reduction. The anion exchange and hydrophobic interaction columns used in the antibody fragment process were shown to have high E. coli DNA clearance capability, with greater than 5.1 and 5.3 logs clearance, respectively. Compared to the large-scale process, higher log reduction values were achieved in small-scale spike clearance studies by challenging the chromatographic steps with load DNA levels 2-5 logs higher than the large-scale process levels. Using highly specific and sensitive spike clearance methods, we demonstrated consistently high DNA clearance factors for each of the production processes that meet industry and regulatory standards for human therapeutics. The method is applicable to a broad range of industrial scale processes where demonstration of the robustness of DNA clearance is necessary to support development or licensure of biopharmaceutical products.