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Mass Spec 2018: Roundtable Session
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The roundtable sessions are truly interactive workshops to connect and discuss real issues with peers. These sessions were designed to be informal (but structured) discussions on topics which are of interest to participants but were not able to be incorporated into the other sessions within the program.

The Mass Spec 2018 roundtable session allowed attendees to interact and discuss the following topics. If you missed a topic that interested you and want to see what was discussed, please use the links below to see the notes.



Table 1: Challenges During Regulatory Submissions

Facilitator: Ramsey Saleem, Amgen Inc.

Scribe: John Valliere-Douglass, Seattle Genetics, Inc.

Scope:
This table will focus on a discussion of effective strategies for presenting mass spectrometry (MS) data and results to regulators in early and late stage filings. The scope of discussion will include phase appropriate MS methods for product characterization, comparability and release testing. We will also discuss approaches for presenting complex MS data sets in a straightforward, succinct manner. Specifically, what level of annotation and detail is sufficient and appropriate given the intended use of the assay in the filing.

To view the table abstract and discussion notes, click here.



Table 2: Process Analytics Technology and Mass Spectrometry

Facilitator: Ananya Dubey Kelsoe, Waters Corporation

Scribe: Nalini Sadagopan, Agilent Technologies

Scope:
Implementation of an efficient, agile, cost effective and flexible biopharmaceutical manufacturing workflow is both a regulatory and industry requirement. Quality of process understanding is key to such an effort. This in turn is dependent on analytical tools that are utilized. Mass Spectrometry hyphenated with high resolution separation platforms can be a powerful tool for real-time process monitoring; the technique has the potential to shrink biologics development cost and timeline. Recent discussions on applying Multi-Attribute assay for process improvement have created heightened awareness in this space.

To view the table abstract and discussion questions, click here.



Table 3: MS in the Biomanufacturing Environment

Facilitator: Hirsh Nanda, Janssen R&D LLC 

Scribe: Sean McCarthy, SCIEX

Scope:
Modern biologics such as monoclonal antibodies are chemically complex and susceptible to numerous biological and chemical modifications during manufacturing, fill/finish and storage. Characterizing and monitoring critical attributes of these compounds during manufacturing can be a significant challenge and requires a battery of assays to ensure batch to batch consistency. High resolution mass spectrometry is an established technique providing molecular level detail in product characterization yet implementation in manufacturing facilities faces technical and regulatory hurdles. 

In this discussion we will cover the challenges of implementing mass spectrometry assays in a manufacturing environment. The potential for deeper product knowledge and more robust processes through in-line testing is driving industry wide efforts to elevate mass-spec assays in manufacturing. New capabilities in MS instrumentation and data analysis are making peptide mapping and intact mass methods more robust and possible for getting MS into QC labs. Non-platform modalities such as ADCs, multi-specifics and vaccines present their own unique challenges. 

To view the table abstract and discussion notes, click here.



Table 4: Host Cell Protein Analysis

Facilitator: Annemiek Verwilligen, Janssen Vaccines & Prevention

Scribe: Sarah Rogstad, CDER, FDA

Scope:
Major hurdles in the detailed analysis of host cell proteins (HCPs) have been lowered by the introduction of mass spectrometry in this field supplying knowledge on protein abundance and identity. Sensitivity increases of LCMS based methods in recent years have subsequently increased HCP data output enormously. Particularly of interest for this topic are best strategies for HCP analysis and best practices for data interpretation and reporting. The focus of this round table will be on recombinant protein and peptide products, whereas viral vaccines will be considered out of scope. As this round table will focus on strategic approaches, overly technical details will be also be considered out of scope. This round table aims to discuss the technical aspects of obtaining, interpreting, and reporting HCP data.

To view the table abstract and discussion notes, click here.



Table 5: Automation

Facilitator: Sreekanth Suravajjala, Amgen Inc.

Scribe: Kristin Boggio, Pfizer, Inc. 

Scope:
In today’s world, where technology driven, smart, and automated machines are arguably improving our everyday lives, it feels that biotech needs to do its part to catch up and provide us some relief. As organizations look to becoming more efficient, automation is a key driver for yielding success. With experiments becoming ever more complex and datasets more immense, the question we often come across is, can we automate biotech? 

Automation is indeed rapidly catching up and playing a significant role in biotech. Today many more tools are being developed that specifically influence analytical workflows. While regular tests such as physical measurements and 1D chromatography have been automated to a high degree, the practical application of automation to mass spectrometry contains multiple opportunities for automation, such as automated sample preparation, the multi-attribute method (MAM), data analysis software, and automated report generation. One of the common automation tools in analytical labs are robotic liquid handlers employed for high-throughput purposes that can also be utilized for automated sample preparation. Another area of automation is using process analytical technology (PAT) to allow for increased process knowledge and apply levers for process control (PAC). Using PAT, organizations have also been able to perform at-line testing and processing for real-time release testing (RTRT). By removing the human component and automating both experimental workflows and data analysis, errors due to human bias and transcription will also be reduced as well. A recent survey article in Nature indicated that the major issue affecting science is reproducibility. In that survey more than 2/3rd of respondents claimed to have tried and failed to replicate another scientist’s work, and what’s stunning is that >1/2 failed to reproduce their own experiments. Researchers believe that one can not only solve the reproducibility conundrum but overall, life sciences can be transformed by automation. Ultimately, such a transformation could boost the overall efficiency and quality of research, helping scientists to complete work and project teams to move faster, and help to accelerate the field of life sciences.

To view the table abstract and discussion notes, click here.



Table 6: In vivo Biotransformation Analysis Strategies and Real Lab Experiences

Facilitator:
Olga Friese, Pfizer, Inc.

Scribe: Yelena Lyubarskaya, Sanofi

Scope:
Peripheral, non-lysosomal catabolism of biotherapeutics is referred to as “biotransformation” and is systemic, with the major sites of enzymatic breakdown by exo- and/or endopeptidases being blood, liver, and kidney. Although clinical safety may not be generally affected by catabolic breakdown of biotherapeutics, the potential impact of circulating catabolites on potency and clearance is unknown. Also, the knowledge of specific sites of proteolytic cleavage is critical for engineering more stable biotherapeutic candidates by mutation of susceptible residues or application of other types of stabilization. Additionally, the protein therapeutics can undergo other in vivo biotransformations such as Asn deamidation, Asp isomerization, Met and Trp oxidation, glycation, N- and C-terminal processing, etc. that might also impact safety, potency, and clearance. In addition, the traditional bioanalytical tools for quantification of biotherapeutics and determining pharmacokinetic (PK) exposure such as ligand-binding assays (LBAs) may not take catabolism/biotransformation into account, and the resulting concentration measures may therefore either underestimate or overestimate true exposure. Therefore, more and more emphasis is put on LC-MS-based analytical tools for both qualitative and quantitative analyses of biotherapeutics in vivo. The goal of this roundtable is to discuss the analytical strategies for in vivo biotransformation analysis of biotherapeutics and provide practical guidance based on the real lab experiences.

To view the table abstract and discussion notes, click here.



Table 7: Comparability

Facilitator:
Karen Lee, Sanofi

Scribe: Jason Rouse, Pfizer, Inc.

Scope:
As described in the ICH Q5E comparability guidance document, biopharmaceutical companies frequently make changes to the manufacturing processes of their respective products both during development and after approval. The reasons for such changes include improving the manufacturing process, increasing scale, adding a new facility, improving product stability, and complying with changes in regulatory requirements. When changes are made to the manufacturing process, the relevant quality attributes (QAs) are evaluated in both pre-change and post-change batches to demonstrate that no significant physicochemical and biological modifications occurred in the active substance that would adversely impact the safety and efficacy of the product. This roundtable aims to discuss the principles and strategic elements of comparability exercises, the practical application of mass spectrometry (MS) in comparability exercises, and how MS characterization endpoints can augment the final demonstration of product comparability.

To view the table abstract and discussion notes, click here.



Table 8: MS/MS Sequencing of Proteins, Do We Need to Do It and When?

Facilitator: Bruno Genet, Sanofi

Scribe: Anders Lund, Synlogic

Scope:
The sequence of a protein has been done for many years by Edman sequencing. The current MS instruments and the MS/MS capabilities are now widely used to verify the sequence. However, “Following ICH Q6B guideline, in appendix 6.1.1 paragraph Structural characterization and confirmation, it is noted that “The amino acid sequence of the desired product should be determined to the extent possible using approaches such as those described in items b) through e) and then compared with the sequence of the amino acids deduced from the gene sequence of the desired product.” with “b) Amino acid composition”, “c) Terminal amino acid sequence”, “d) Peptide map” and “e) Sulfhydryl group(s) and disulfide bridges”. The term “sequencing” must be discussed and clarified in order to fit with the authorities’ expectations.

To view the table abstract and discussion questions, click here.



Table 9: Characterization of Antibody Drug Conjugates (ADCs)

Facilitator: Midori Greenwood-Goodwin, Genentech, a Member of the Roche Group

Scribe: Sunnie Kim, Seattle Genetics, Inc.

Scope:
Antibody-drug conjugates (ADCs) represent a unique class of drugs consisting of a monoclonal antibody (mAb), cytotoxic small molecule and a stable chemical linker. LC-MS is routinely used to quantify and characterize the physicochemical properties of ADCs. LC-MS is further used throughout the drug development process to assess small molecule payload, the ratio of drug to antibody and the presence of naked antibody compared to all ADCs. The level of complexity and heterogeneity associated with ADCs presents unique challenges to traditional mAb characterization methods. The level of characterization is typically dependent on the phase of the program and the criticality of the product attribute that’s being monitored. This roundtable aims to discuss both the technical and strategic challenges associated with ADC characterization. 

To view the table abstract and discussion notes, click here.



Table 10: Forced Degradation

Facilitator: Tom Chen, Bayer

Scribe: Jason Gilmore, Seattle Genetics, Inc.

Scope:
Forced degradation studies are used to characterize prospective drug substance/drug product under extreme conditions and can be used to guide candidate molecule selection, analytical method development, formulation development, and comparability studies. It is required to demonstrate specificity of stability indicating methods and provides an insight into degradation pathways and degradation products of the drug substance. At present, regulatory guidance is very general and does not fully define the design and implementation of forced degradation studies. In this session, we will discuss the scope of forced degradation studies and the appropriate application of mass spectrometry to characterize product quality changes and degradation pathways that become evident under stress conditions.

To view the table abstract and discussion notes, click here.



Table 11: Deep Dive into Genesis of Trisulfide Formation and Analysis By MS

Facilitator: Zhiqi Hao, Genentech, a Member of the Roche Group

Scribe: Delia Li, Genentech, a Member of the Roche Group

Scope:
Trisulfides are variants of disulfides formed by the insertion of a sulfur atom into a disulfide bond. They are common post-translational modifications found in interchain linkages in recombinant and natural antibodies, as well as in nonclinical preparations and commercial therapeutics. In recombinant therapeutics, they are believed to be generated during cell culture from the reaction of an intact disulfide bond with dissolved hydrogen sulfide (H2S). While their biological function is still largely unknown, trisulfides may potentially impact protein structure and function and have also been found to introduce heterogeneity that can directly affect antibody-drug conjugate (ADC) generation. As such, sensitive and reliable analytical approaches are necessary for monitoring and controlling trisulfide levels in process development and in products. This roundtable will focus on mass spectrometry-based techniques for trisulfide identification/quantification, their challenges, and their application to understanding the impact of cell culture process conditions on trisulfide formation.

To view the table abstract and discussion notes, click here.



Table 12: Intact Analysis/Top Down Analysis

Facilitator: Srikanth Kotapati, Bristol-Myers Squibb Company

Scribe: David Passmore, RubrYc Therapeutics Inc.

Scope:
Mass spectrometry provides critical information for the characterization and identification of proteins. Top down analysis is increasingly being preferred over conventional peptide based and sub-fragment based approaches of protein characterization, since the protein is introduced into the mass spectrometer in its “native” state. However, there are several challenges associated with top down analysis such as protein separation, sensitivity and data complexity. This roundtable aims to discuss the applications, challenges and recent advances in Intact/Top Down Analysis.

To view the table abstract and discussion notes, click here.



Table 13: System Suitability Strategies for Mass Spectrometers and LC/MS Methods

Facilitator: Ying Zhang, Pfizer, Inc.

Scribe: Frances Namuswe, CDER, FDA

Scope:

System suitability testing is an integral part of many analytical procedures including mass spectrometry analysis. Lack of evaluating LC/MS systems prior to analyzing samples may generate data with poor quality, which could potentially mislead data interpretation and possibly cause extensive investigations and delays in the development timelines for biotherapeutic products. This roundtable aims to discuss different aspects of system suitability in LC/MS methods as applied to in-depth characterization of biotherapeutic products. Topics include approaches and strategies for determining system suitability samples for different LC/MS methods, confirming instrument performance, and ensuring reproducible and accurate results for regulatory submission. System suitability for MS in QC (i.e., multi-attribute method) will also be discussed.

To view the table abstract and discussion notes, click here.



Table 14: Challenges for Bi-specifics/Tri-specifics

Facilitator: Greg Staples, Agilent Technologies

Scribe: Nicole Liu, Genentech, a Member of the Roche Group

Scope:
The interest in bispecific and trispecific antibodies is increasing for therapeutic applications, and there is a growing list of molecules currently in clinical studies. These multispecific molecules are capable of binding more than one antigen, and as a class of molecules comprise a large number (more than 50) of different molecular formats. For example, some molecules resemble typical IgG structure while others lack an Fc region. In general, bi- and tri-specific antibodies are assembled from antigen-binding and dimerization building blocks to form the intended functional therapeutic. However, these building blocks can be combined in a number of ways, resulting in closely related but ultimately undesired end-product molecules. These impurities may be very different in mass (e.g. half antibodies) or very close in mass (e.g. light-chain scramble). Consequently, the analysis of bi- and trispecific antibodies using mass spectrometry (MS) presents unique and significant challenges. In this roundtable discussion, we will take a deep dive into the use of MS for bi- and trispecific antibody analysis, seeking to build consensus by identifying trends from participants’ success stories, as well as identifying opportunities from participants’ ongoing challenges.

To view the table abstract and discussion notes, click here.



Table 15: Software

Facilitator: Joe Shambaugh, Genedata, Inc.

Scribe: Brian Gau, Pfizer, Inc.

Scope:

The biologics discovery and development process has become increasingly complex over recent years. Meanwhile, advances in analytical technologies continues to create ever-increasing amounts of highly complex data. Such data is typically generated in multiple locations using different instruments over several years and exists in a number of formats. Various software solutions are employed to analyze, manage and archive data and results.

This round table will discuss the software currently deployed within organizations as well as data analysis challenges, bottlenecks, and best practices. The conversation will start by having each participant describe their use of software with respect to the various areas that are listed in the round table topics. The goal will be to identify commonalities as well as gaps.

To view the table abstract and discussion notes, click here.


 

 

 

 

 

 

 

 


 





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