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Late Afternoon Session
Joint Plenary Session – Formulation, Aggregation and Stability Assessment
Residual host cell proteins (HCP) are one of the critical quality attributes (CQAs) for biotherapeutic products, and as such require identification and quantitation. Gel-free, label-free mass spectrometry (MS) is an orthogonal method to ELISA, and has an invaluable role in characterization of HCPs. We have established robust workflows for the characterization, monitoring and quantitation of low level HCPs by LC-MS/MS. For identification and relative quantification of HCPs, samples are subjected to trypsin digestion, fractionation to reduce the complexity of the tryptic peptide mix due to the presence of the DS in very high amounts, and high mass accuracy shotgun RPLC-MS/MS. PQCs (Process Quality Controls) are analysed alongside the samples as a measure of the instrument and analytical procedure performance. HCPs are identified by using an unbiased bioinformatics workflow with multiple search engines to match detected peptides against a custom process-specific HCP database (i.e., CHO, human, yeast and bacteria), and spectral counts provide a qualitative assessment of relative abundance of each HCP. The sensitivity of the method depends on the particular characteristics of the DS, but has been shown to reproducibly identify spiked proteins in the presence of drug substance between 1-10 ppm. For absolute quantitation of HCPs, a multiplexed targeted LC-MRM/MS assay is developed using stable-isotope labelled peptides representing the specific HCPs to be monitored. QC samples and calibration curves are run alongside study samples, and stable-isotope labelled peptides are spiked into all samples as internal standards. The sensitivity of detection is routinely achieved in the 1 ppm range. Examples will be presented from mAbs and recombinant proteins, including monitoring HCP profiles to assess process changes, improvements or scale-up, to demonstrate batch uniformity, to understand clearance. We will also present examples comparing HCP profiles of Biosimilars vs Innovators.
Late Morning Session
As the biopharmaceutical industry sets its sights on developing ever-more stucturally complex molecules, there is a growing requirement for complementary analytics to ensure detailed and robust characterization profiles are generated.
Here, we will demonstrate two case studies where single-pass analytics proved insufficient in elucidating and understanding protein behavior at a molecular level. We’ll discuss the cases of small 2-domain recombinant protein contructs, and also biosimilar molecules, which both needed characterization using a variety of analytical technologies in order that a complete picture of their stability profiles was revealed
Early Afternoon Session
Direct measurement of biomolecular interactions plays an important role in therapeutic drug discovery and development. Label-free analytical technologies, provide a powerful tool to obtain accurate information about the kinetics of biomolecular complex formation and its stability. Both are key components of characterizing a drug-target interaction.
Real time data on specificity, affinity, kinetics of binding and mapping of if binding sites ensures the selection of the best candidates. High throughput and efficient selection of therapeutic candidates early in the drug development process handling challenging matrixes like crude biologicals or fragments in DMSO will save time and resources and prevent late-stage failures. The session will cover the use of BLI technology for biotherapeutics screening, inhibitor screening and cell based approaches. We will also present a very innovative approach to SPR, using dynamic injection (di-SPR) methods to decrease “time to hit” ratio and assess ligand complementarity when working with fragments and small molecules.
Several methods for glycoanalysis of antibodies are presented. This includes chromatographic, electrophoretic and mass spectrometry based methods. A focus will be on high-throughput techniques. A study where we compared the results for a therapeutic antibody obtained with different methods will be shown. The influence of glycosylation on function of antibodies will be included.
With the rise of new therapeutic formats such as gene and cellular therapies, the need for robust bioanalytical methods is increasing.