Mass Spec 2020

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.

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.

Changes in cellular protein concentrations and isoform expression are dictated by transcriptional, translational, and protein degradation rates and can reflect disease processes. Protein post-translational modifications (PTMs), especially those related to disease processes, add layers of potential functional modulation. Capturing disease proteomic signatures in body fluids like plasma or dried blood may provide insights into underlying disease mechanisms and potential avenues for diagnostic, prognostic, and progression biomarkers. Underlying precision health centers individual’s proteomic signature, especially those which evolve over time will provide physicians with clinically actionable diagnosis. With enhanced adoption of telehealth with the COVID-19 pandemic could increase the integration of semi continuous biochemical blood-based monitoring into the US health care system. Blood components include resident plasma proteins, and proteins from tissue leakage which circulate in response to systemic stimuli and include innate immunity (e.g. cytokine and others). With large scale proteomic analysis of 100-1000s of samples, the biological, environmental, and genetic impact on health and disease signatures should underscore mechanistic and biological variability. We have developed a high throughput robust pipeline from remote sample procurement, shipping, automated sample processing and LC-MSMS methods for targeted discovery and selective pathways (e.g. acute phase). The application of multiple modal pipeline also accommodates for plasma. We deployed remote sampling device to track individuals with COVID-19 as well as patients with mid-risk cardiac diseases and other situations. The application of mass spectrometer from discovery to use in a clinical chemistry lab is essential to this transition. We will discuss the process and quality control behind the sciences in allowing the application of remote sampling into clinical situation. The promise of proteomics and the use of protein-based mass spectrometry in the clinical domain has been widely stated, and although adopt still has a number challenges process for success are being put into place for its operationalization.

Changes in cellular protein concentrations and isoform expression are dictated by transcriptional, translational, and protein degradation rates and can reflect disease processes. Protein post-translational modifications (PTMs), especially those related to disease processes, add layers of potential functional modulation. Capturing disease proteomic signatures in body fluids like plasma or dried blood may provide insights into underlying disease mechanisms and potential avenues for diagnostic, prognostic, and progression biomarkers. Underlying precision health centers individual’s proteomic signature, especially those which evolve over time will provide physicians with clinically actionable diagnosis. With enhanced adoption of telehealth with the COVID-19 pandemic could increase the integration of semi continuous biochemical blood-based monitoring into the US health care system. Blood components include resident plasma proteins, and proteins from tissue leakage which circulate in response to systemic stimuli and include innate immunity (e.g. cytokine and others). With large scale proteomic analysis of 100-1000s of samples, the biological, environmental, and genetic impact on health and disease signatures should underscore mechanistic and biological variability. We have developed a high throughput robust pipeline from remote sample procurement, shipping, automated sample processing and LC-MSMS methods for targeted discovery and selective pathways (e.g. acute phase). The application of multiple modal pipeline also accommodates for plasma. We deployed remote sampling device to track individuals with COVID-19 as well as patients with mid-risk cardiac diseases and other situations. The application of mass spectrometer from discovery to use in a clinical chemistry lab is essential to this transition. We will discuss the process and quality control behind the sciences in allowing the application of remote sampling into clinical situation. The promise of proteomics and the use of protein-based mass spectrometry in the clinical domain has been widely stated, and although adopt still has a number challenges process for success are being put into place for its operationalization.

The Blaze system directly couples high-resolution MS analysis with imaged capillary isoelectric focusing (iCIEF) to enable monitoring of multiple critical quality attributes in a single 15-minute iCIEF-MS assay. In this Technical Seminar, we will present details of the Blaze system’s design, including a novel nebulizer chip design. Detailed characterization of charge variants by chip-based electrospray ionization and interfacing to multiple MS systems including SCIEX, Thermo and Bruker will be presented. Comparative data on the Blaze system compared to legacy cIEF will also be shown. A new, streamlined software workflow from Protein Metrics for Blaze will demonstrate the combined analysis of iCIEF and MS data. The latest results from our biopharmaceutical collaborators demonstrate use of Blaze to monitor a wide variety of mAb structural variants.