Keynote Session I: Advantages of Emergent MS Methods for Analysis of Clinically Relevant Glycans

Sep 14, 2020 7:00am ‐ Sep 14, 2020 8:15am

The heterogeneity and fragility of free glycans and the carbohydrate modifications to proteins and lipids present challenges for their analysis by mass spectrometry, but new instrumentation, accessories, and protocols can successfully address these challenges. Novel sample preparations, new ionization and scanning modes, and Electron-Based Dissociation (ExD) options offer opportunities to preserve labile modifications to biopolymers and provide both glycosidic and cross-ring cleavage information on glycans, on a time scale that is compatible with online nanoflow liquid chromatography and capillary electrophoresis, alone or in combination with ion mobility separations. Examples poised to meet expanding clinical and industry needs will include emergent methods that are already in use for medical studies, or are ready to move from our own or other research laboratories to the clinical laboratory.

Keynote Session I: Advantages of Emergent MS Methods for Analysis of Clinically Relevant Glycans

Sep 14, 2020 7:15am ‐ Sep 14, 2020 8:15am

The heterogeneity and fragility of free glycans and the carbohydrate modifications to proteins and lipids present challenges for their analysis by mass spectrometry, but new instrumentation, accessories, and protocols can successfully address these challenges. Novel sample preparations, new ionization and scanning modes, and Electron-Based Dissociation (ExD) options offer opportunities to preserve labile modifications to biopolymers and provide both glycosidic and cross-ring cleavage information on glycans, on a time scale that is compatible with online nanoflow liquid chromatography and capillary electrophoresis, alone or in combination with ion mobility separations. Examples poised to meet expanding clinical and industry needs will include emergent methods that are already in use for medical studies, or are ready to move from our own or other research laboratories to the clinical laboratory.

Technical Seminar Sponsored by Thermo Fisher Scientific: Quantification of Common and Troublesome CHO-HCPs using a Novel Multipoint SureQuant Panel

Sep 14, 2020 8:15am ‐ Sep 14, 2020 8:45am

The quantification of host cell proteins (HCPs) during process development of therapeutics remains an important topic to understand and optimize conditions for their removal. HCPs can often lead to dangerous and deleterious effects on produced therapeutics. Mass spectrometry-based methods can offer high specificity and accurate determination of HCPs quantitation with respect to the standard ELISA based techniques. Herein, we developed a novel HCP quantitation method based on Thermo Fisher Scientific’s SureQuant Targeted Quantitation Assay, a technique that delivers heavy labelled tryptic peptides for targeted quantitation. A panel of 282 peptides was developed to target common and troublesome HCPs in Chinese Hamster Ovary cell lines (CHO), and the panel was subsequently implemented to analyze vaccine and monoclonal antibody therapeutics during process development.

Session I – Molecular Design, Developability and Biotransformation

Sep 14, 2020 8:45am ‐ Sep 14, 2020 9:50am

Introduction

Sep 14, 2020 10:05am ‐ Sep 14, 2020 10:10am

Session II – Product Characterization and Comparability

Sep 14, 2020 10:05am ‐ Sep 14, 2020 11:40am

Regulatory Considerations for Multi-Attribute Method Implementation

Sep 14, 2020 11:10am ‐ Sep 14, 2020 11:40am

Mass spectrometry based multi-attribute method (MAM) has been commonly proposed to be implemented as a quality control (QC) method for monitoring product critical quality attributes (CQA) of biotherapeutics. Different from characterization methods, regulatory requirements should be followed for developing a QC method for release and stability testing. However, there are some method specific issues for MAM as an advanced technology, for example the additional considerations for method validation and new peak detection feature based on the intended use. Because the ultimate implementation goal of MAM is to replace and consolidate several conventional methods, such as Cation Exchange Chromatography (CEX) for the control of charge variants and Hydrophilic Interaction Chromatography (HILIC) for the control of glycan variants, additional regulatory considerations should also be addressed before its final implementation, for example the comparison between MAM and conventional methods and product specific risk assessment. In this presentation, major regulatory points-to-consider and relevant data expectation to support MAM implementation will be discussed with several case studies along with strategies of rolling in MAM at an appropriate stage during product development.

Keynote Session II: Spontaneous Modifications in Long-Lived Proteins: Structural and Biological Implications

Sep 15, 2020 7:00am ‐ Sep 15, 2020 8:05am

Long-lived proteins such as alpha-synuclein, A-beta, and tau are subject to a variety of spontaneous chemical modifications. These modifications accumulate over time without the need for enzymatic catalysis. Isomerization at specific amino acid residues, including aspartic acid and serine, represents one of the more abundant spontaneous chemical modifications and one of the least studied and most difficult to detect. Indeed, traditional proteomics experiments are completely blind to such sites of isomerization. We demonstrate that isomerized residues significantly perturb proteolysis by cathepsins. In fact, residual peptide fragments of 3-4 amino acids persist even after treatment with both endo- and exopeptidases. The rates of degradation are also significantly slowed, in addition to reduced cleavage sites. We further show that isomerization takes place on a timescale of weeks, significantly increasing the pool of potentially contributing proteins. The residual fragments, which still include an unnatural amino acid, are unlikely to be suitable substrates for transporter recognition and release from the lysosome. These observations provide the foundation for a novel class of substrate-induced lysosomal malfunction that may be related to lysosomal storage associated with many age-related disorders.

Keynote Session II: Spontaneous Modifications in Long-Lived Proteins: Structural and Biological Implications

Sep 15, 2020 7:05am ‐ Sep 15, 2020 8:05am

Long-lived proteins such as alpha-synuclein, A-beta, and tau are subject to a variety of spontaneous chemical modifications. These modifications accumulate over time without the need for enzymatic catalysis. Isomerization at specific amino acid residues, including aspartic acid and serine, represents one of the more abundant spontaneous chemical modifications and one of the least studied and most difficult to detect. Indeed, traditional proteomics experiments are completely blind to such sites of isomerization. We demonstrate that isomerized residues significantly perturb proteolysis by cathepsins. In fact, residual peptide fragments of 3-4 amino acids persist even after treatment with both endo- and exopeptidases. The rates of degradation are also significantly slowed, in addition to reduced cleavage sites. We further show that isomerization takes place on a timescale of weeks, significantly increasing the pool of potentially contributing proteins. The residual fragments, which still include an unnatural amino acid, are unlikely to be suitable substrates for transporter recognition and release from the lysosome. These observations provide the foundation for a novel class of substrate-induced lysosomal malfunction that may be related to lysosomal storage associated with many age-related disorders.

Technical Seminar Sponsored by SCIEX: Are Off-The-Shelf Formulation Buffers Suitable for Your Biotherapeutic Protein? The Effect of Buffer Formulations on Antibodies’ Proteoforms Relative Abundances

Sep 15, 2020 8:05am ‐ Sep 15, 2020 8:35am

A number of buffers, approved by the FDA/EMA, are sold as being suitable for the formulation of antibody therapeutics. These buffers cover a wide range of substances, pH, salts, amino acids, sugars, and detergents.

Some of them have been optimized for certain antibodies in particular. Typically, pre-formulation of Abs starts at these sets of buffers, followed by further improving the conditions of a few selected buffers thereafter. However, to our knowledge, there is no study that has assessed the effect of these buffer formulations on the relative amounts of the antibody modifications (i.e., glycosylation, oxidation and others). Here we present a multiple attribute methodology (MAM) workflow on intact proteins upon reconstruction of data (intact MAM) using liquid chromatography coupled to high resolution mass spectrometry (LC-MS). For this study the effects of 96 commercially available buffer formulations were assessed focusing on the relative abundances of endogenous modifications of the Trastuzumab and our inhouse antibody 17b. For the evaluation, an LC-MS method and data processing workflow for intact MAM were established. Results are presented for a time course study of 7 days of storage at multiple temperatures determining the effect of the different formulations with the optimized method enabling the throughput needed.