Report of the international conference on manufacturing and testing of pluripotent stem cells.

Sessions included an overview of past cell therapy (CT) conferences sponsored by the International Alliance for Biological Standardization (IABS). The sessions highlighted challenges in the field of human pluripotent stem cells (hPSCs) and also addressed specific points on manufacturing, bioanalytics and comparability, tumorigenicity testing, storage, and shipping. Panel discussions complemented the presentations. The conference concluded that a range of new standardization groups is emerging that could help the field, but ways must be found to ensure that these efforts are coordinated. In addition, there are opportunities for regulatory convergence starting with a gap analysis of existing guidelines to determine what might be missing and what issues might be creating divergence. More specific global regulatory guidance, preferably from WHO, would be welcome. IABS and the California Institute for Regenerative Medicine (CIRM) will explore with stakeholders the development of a practical and innovative road map to support early CT product (CTP) developers.

Industry view on the relative importance of "clonality" of biopharmaceutical-producing cell lines.

Recently, several health authorities have requested substantial detail from sponsor firms regarding the practices employed to generate the production cell line for recombinant DNA-(rDNA) derived biopharmaceuticals. Two possible inferences from these regulatory agency questions are that (1) assurance of "clonality" of the production cell line is of major importance to assessing the safety and efficacy of the product and (2), without adequate proof of "clonality", additional studies of the cell line and product are often required to further ensure the product's purity and homogeneity. Here we address the topic of "clonality" in the broader context of product quality assurance by current technologies and practices, as well as discuss some of the relevant science and historical perspective. We agree that the clonal derivation of a production cell line is one factor with potential impact, but it is only one of many factors. Further, we believe that regulatory emphasis should be primarily placed on ensuring product quality of the material actually administered to patients, and on ensuring process consistency and implementing appropriate control strategies through the life cycle of the products.

Analytical lessons learned from selected therapeutic protein drug comparability studies.

The successful implementation of process and product changes for a therapeutic protein drug, both during clinical development and after commercialization, requires a detailed evaluation of their impact on the protein's structure and biological functionality. This analysis is called a comparability exercise and includes a data driven assessment of biochemical equivalence and biological characterization using a cadre of analytical methodologies. This review focuses on describing analytical results and lessons learned from selected published therapeutic protein comparability case studies both for bulk drug substance and final drug product. An overview of the currently available analytical methodologies typically used is presented as well as a discussion of new emerging analytical techniques. The potential utility of several novel analytical approaches to comparability studies is discussed including distribution and stability of protein drugs in vivo, and enhanced evaluation of higher-order protein structure in actual formulations using hydrogen/deuterium exchange mass spectrometry, two-dimensional nuclear magnetic resonance fingerprinting or empirical phase diagrams. In addition, new methods for detecting and characterizing protein aggregates and particles are presented as these degradants are of current industry-wide concern. The critical role that analytical methodologies play in elucidating the structure-function relationships for therapeutic protein products during the overall assessment of comparability is discussed.

Health Canada/BIOTECanada Summit on regulatory and clinical topics related to subsequent entry biologics (biosimilars), Ottawa, Canada, 14 May 2012.

In May 2012, Health Canada and other participants held a National Summit on Subsequent Entry Biologics (SEBs). Health Canada released a guidance document in March 2010 describing policy positions and data requirements for approval of SEBs. While Health Canada and health agencies in other regulatory jurisdictions are aligned on many scientific principles related to biosimilar drugs, Health Canada's specific requirements may not be widely understood by many Canadian stakeholders. The Summit provided an opportunity for education and dialog among physicians who prescribe biologics, provincial payers, and industry on the following topics: preclinical and clinical comparability studies; manufacturing and other product differences; extrapolation of indications; substitution and interchangeability of SEBs with reference biologic drugs in clinical practice; payers' current perspective; pharmacovigilance and naming. It is anticipated that the consensus reached at this meeting will further educate Canadian healthcare professionals, provincial payers, and insurers about the appropriate use of SEBs, and may be of general interest to others internationally.

Comparability assessments of process and product changes made during development of two different monoclonal antibodies.

To assess the impact of manufacturing changes on antibody structure and function during the course of product development, three comparability studies were performed for each of two different IgG1 monoclonal antibody product candidates. Comparability study #1 evaluated the effect of changing the cell line and bulk drug substance manufacturing process for cell culture and purification. Results indicated that these process changes led to differences in sialylation of N-glycans and/or C-terminal lysine levels. Comparability study #2 results confirmed that scale-up of the bulk process and transfer to the commercial site, combined with changing from a lyophilized to a liquid dosage form, did not impact the structural or functional integrity of the antibodies. Comparability study #3 examined possible differences arising when the liquid formulation filled into pre-filled syringes and vials. Results indicated nearly identical molecular structure, biological activity, and degradation profiles except for a small yet statistically significant increase in the levels of subvisible particles in pre-filled syringes. These results from comparability studies with two different monoclonal antibodies are discussed with respect to the timing of the manufacturing changes and overall comparability strategies to assure safety and efficacy during development.

The Science is There: Key Considerations for Stabilizing Viral Vector-Based Covid-19 Vaccines.

Once Covid-19 vaccines become available, 5-10 billion vaccine doses should be globally distributed, stored and administered. In this commentary, we discuss how this enormous challenge could be addressed for viral vector-based Covid-19 vaccines by learning from the wealth of formulation development experience gained over the years on stability issues related to live attenuated virus vaccines and viral vector vaccines for other diseases. This experience has led -over time- to major improvements on storage temperature, shelf-life and in-use stability requirements. First, we will cover work on 'classical' live attenuated virus vaccines as well as replication competent viral vector vaccines. Subsequently, we address replication deficient viral vector vaccines. Freeze drying and storage at 2-8 °C with a shelf life of years has become the norm. In the case of pandemics with incredibly high and urgent product demands, however, the desire for rapid and convenient distribution chains combined with short end-user storage times require that liquid formulations with shelf lives of months stored at 2-8 °C be considered. In confronting this "perfect storm" of Covid-19 vaccine stability challenges, understanding the many lessons learned from decades of development and manufacturing of live virus-based vaccines is the shortest path for finding promising and rapid solutions.

PDA/FDA Adventitious Agents and Novel Cell Substrates: Emerging Technologies and New Challenges, Nov. 3-4, 2011, Rockville, MD.

In response to the challenges of addressing potential safety concerns related to the use of novel cell substrates, the Parenteral Drug Association (PDA) and the U.S. Food and Drug Administration (FDA) co-sponsored a conference on November 3-4, 2011 in Rockville, Maryland, USA. The following were discussed: the current adventitious agent testing methods and the use of emerging nucleic acid-based technologies for broad detection of known and novel viruses; issues associated with manufacture of biologicals in novel cell substrates such as insect cell lines and plants, with specific focus on potential safety concerns related to endogenous retroviral elements; and raw material controls and strategies to mitigate risk of adventitious virus contamination during product manufacture.

Evolution of approaches to viral safety issues for biological products.

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA) Approaches to viral safety issues for biological products have evolved during the past 50+ years. The first cell culture products (viral vaccines) relied largely on the use of in vitro and in vivo virus screening assays that were based upon infectivity of adventitious viral agents. The use of Cohn fractionation and pasteurization by manufacturers of plasma derivatives introduced the concepts that purification and treatment with physical and chemical agents could greatly reduce the risk of viral contamination of human albumin and immunoglobulin products. But the limitations of such approaches became clear for thermolabile products that were removed early in fractionation such as antihemophilic factors, which transmitted hepatitis viruses and HIV-1 to some product recipients. These successes and limitations were taken into account by the early developers of recombinant DNA (rDNA)-derived cell culture products and by regulatory agencies, leading to the utilization of cloning technology to reduce/eliminate contamination due to human viruses and purification technologies to physically remove and inactivate adventitious and endogenous viruses, along with cell banking and cell bank characterization for adventitious and endogenous viruses, viral screening of biological raw materials, and testing of cell culture harvests, to ensure virus safety. Later development and incorporation of nanofiltration technology in the manufacturing process provided additional assurance of viral clearance for safety of biotechnology products. These measures have proven very effective at preventing iatrogenic infection of recipients of biotechnology products; however, viral contamination of production cell cultures has occasionally occurred. Screening tests for viral contamination in raw materials have not proven practical by themselves to prevent contamination of cell cultures, but they can be made more effective by coupling with treatment using physical or chemical agents to further reduce the hypothetical viral loads present in cell culture raw materials. Recent advances in polymerase chain reaction (PCR) technology have allowed preharvest testing for specific viral agents to reduce the risk of cell culture contamination by specific viruses in the harvest material. Examples of each of these stages in the evolution of virus detection methods are described and assessed in this paper.

Summary of breakout session e.

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA).

Apparent virus contamination in biopharmaceutical product at centocor.

Out-of-specification (OOS) results were reported by a contract lab in the in vitro adventitious agent assay (AVA) for two products manufactured using mouse myeloma cells in perfusion bioreactors. Cytopathic effect observed for test article-inoculated MRC-5 monolayers resembled foci seen in tissue culture cells infected with transforming viruses. All reasonable known technologies, including highly sensitive, state-of-the-art methodologies and multiple, redundant, and orthogonal methods, were deployed to screen broadly for potential viral and microbial contaminants. Due to the appearance of apparent foci, testing for murine, bovine, and human polyomavirus contamination was heavily represented in the analytical investigation. The results obtained in this extensive screening provided convincing evidence for the lack of an infectious viral or other biological agent. Although the initial investigation produced no reason to invalidate AVA yielding OOS results or to suspect an assay artifact, an extended evaluation revealed several irregularities at the contract test lab reporting the OOS results. The extended investigation also included attempts to reproduce OOS results at alternate contract testing labs and an inter-laboratory study in which methodological differences in the AVA at the three different contract labs were investigated. Only the contract lab initially reporting the OOS results reported foci during this extended evaluation. The results of the inter-laboratory study suggested that the foci artifact might be attributed to the prolonged exposure of the MRC-5 monolayer to cell debris present in the test article. Confocal immunofluorescence microscopy and transmission electron microscopy were subsequently used to provide convincing evidence that the foci observed in test article-inoculated AVA wells were composed of a core of degraded myeloma cell debris covered by one or more layers of MRC-5 cells. The observation that the foci were detected in the AVA at a contract lab where the MRC-5 monolayer is exposed to production cell line debris for a prolonged period strongly suggests that these foci form when MRC-5 grow over the cell debris present in the test article. The cumulative results of the investigation supported the conclusion that the OOS results were artifacts of the AVA test system and not a result of contamination with a virus or other biological agent. Testing was discontinued at the contract lab generating the OOS results and validated at a second contract lab. Manufacturing resumed in consultation with health authorities. The lots were retested following a standard operating procedure (SOP) already in place and ultimately dispositioned for use in normal distribution channels.