Host cell proteins (HCPs) are process-related impurities that must be adequately cleared from recombinant biopharmaceuticals during the downstream process to ensure product quality, purity, and patient safety. It is well known that some HCPs with very little abundance have a strong impact on product quality. Understanding how HCPs escape the purification process and the detection of those HCPs is a prerequisite for developing a suitable HCP depletion process. Here, we assess our research on methods for detection of low abundant HCPs by MS/MS and compare 3 different methods (Hexapeptide-, partial digest, and immunoaffinity-chromatography) to enrich HCP levels in samples.

Monoclonal antibodies consist of a mixture of different proteoforms (e.g. glycoforms) with potentially different functionalities. mAbs activate the immune response via binding to Fc receptors. Therefore, it is important to study their binding to individual mAb proteoforms. Common approaches, such as SPR, provide an overall affinity response for all mAb proteoforms. In this presentation, we want to show an innovative approach based on sheathless CE-MS to study relative affinities of different mAb proteoforms in mixtures towards the FcRn and FcyRIIa receptors. Overall our approach has the capability to boost the study of interactions in a proteoform-resolved manner.

Chemical crosslinks identified by mass spectrometry have been widely applied to study the architectures of protein complexes and networks even in organelles and whole cells. The quantification of crosslinks will provide information on the mechanism of protein complexes like binding affinities and interfaces. Capturing changes in protein interactions in samples like biopsies will aid understanding the molecular basis of diseases at the system level and the classification of disease types.

To meet challenges in host cell protein (HCP) analysis during drug development, especially downstream process development, which entails fast turnaround time and robustness while identifying high level of HCPs and their clearance trend for further purification development, we have developed HCP-automated iterative MS (HCP-AIMS): It is a simple, automated, and robust HCP workflow with deep and unbiased identification and relative quantification capability. This HCP-AIMS approach only requires easy direct digestion of the samples without enrichment or pre-treatment. With the fully automated precursor ion exclusion in MS/MS mode, low-abundance HCP peptides could be selected for MS/MS analysis in iterative replicates.

With the introduction of combined modalities like bispecifics, ADCs, and fusion proteins the complexity of biotherapeutics has experienced a significant increase over the last decades. Herewith associated, the risk of adverse events in patients becomes a critical safety aspect. Understanding the mechanisms, which underlie immunogenicity of biotherapeutics, is a key component for earlier identification, monitoring, and counteracting of such liabilities. Our study provides an in-depth analysis of cross-related factors that govern the formation of ADA-driven immune complexes. Besides, a critical evaluation of analytical methodology will be provided.

The development of miniaturized systems with automation of analytical methods represents an attractive approach to achieve a better understanding of cell culture processes. In this context, we developed a novel microfluidics-based modular system to be coupled to a mAb-producing mammalian cell bioreactor aiming at performing (1) multiplexed immunodetection of relevant proteins, (2) evaluation of glycosylation profile of the target mAb, and (3) analysis of amino acids using microchip capillary electrophoresis.

Time-resolved intrinsic-fluorescence-lifetime extraction (TRIFLE) is an emerging analytical technology for quantifying product and impurities in real-time. TRIFLE can be used to identify and quantify biophysically-similar proteins even when there is overlap in their chromatographic elution profiles. I will discuss examples of TRIFLE being used to identify product and product-related impurities for different modalities, including mAbs and viral vectors, and I will introduce our work on another analytical technology: virus lasers.

A significant burden in biomanufacturing is the characterization of glycans and glycopeptides on biopharmaceuticals. To address the need for customized software to handle multi-attribute data arising from combinations of liquid chromatography, ion mobility, and mass spectrometry (LC-IM-MS), two semi-automated software programs for released glycan and glycopeptide characterization and quantitation are described. Both programs can identify glycans/glycopeptides more accurately and quicker compared to other methods.

Glycine as a signal-boosting additive solves the dilemma between excellent peak separation and sufficient MS sensitivity for glycopeptide analysis using regular-flow ion-paring HILIC-MS. Here we present showcases where this platform was applied to support early and late stages of biopharmaceutical development, including quick glycosylation profiling of monoclonal antibody and fusion proteins, discovery of atypical glycosylation sites of low occupancy in monoclonal antibody, and targeted-based subclass-specific IgGs N-glycosylation profiling in serum.  

Multi-attribute monitoring (MAM) by LC-MS has gained increased interest by Biopharma companies to replace multiple traditional assays in the control system. In this presentation, we will provide Roche/Genentechs perspective on the practical considerations and strategies on MAM use in quality control and replacement of traditional assays, including target attribute selection, new peak detection for stability studies, and bridging strategies to justify traditional assays replacement with MAM.

The quantification of individual host cell proteins (HCP) is a major analytical challenge that bottom-up proteomics workflows start tackling. The implementation of new standards for robust and accurate quantification of HCP will be presented. The potentialities of Data Independent Acquisition coupled with ion mobility separation will be assessed on various latest-generation instrumental platforms. Finally, benefits of using innovative artificial intelligence algorithms for data processing will be highlighted.

Product quality is controlled at all stages of the manufacturing process, at release, and on stability to define the shelf life. Criticality assessment of each product quality attribute provides the basis for defining the required level of control. By combining available knowledge of the molecule’s degradation behavior with understanding how the individual steps of the manufacturing process impact critical quality attributes, a smart control strategy can be developed allowing for manufacturing flexibility without compromising quality and safety of the product.