Miltenyi Biotec 
Cell Therapy 
Analytics Day 2021

Quality control of CD19 CAR T cells – establishment and challenges

Speaker: Regina Gary
Universitätsklinikum Erlangen, Germany

Abstract
The quality control of cellular therapeutics is especially challenging as there is often a huge biological diversity in the cellular composition of peripheral blood of different individuals and, additionally, cells of patients might be affected by medication and disease when compared to healthy donors.Crucial specifications for the release of cell products include (i) that the identity of the cells (here CAR transduced CD3+ T cells) must be confirmed, (ii) the microbiological quality must be proven as cell products are intrinsically not amenable to final sterilization, and (iii) the quality of the cells themselves must be good to optimal (e.g. high vitality).The most important tools for the analysis of cell products are flow cytometry and the different kinds of microbiological testing such as microbiological culture, PCR for the absence of mycoplasma, and endotoxin testing. One of the challenges is the short shelf-life of not-cryopreserved, “fresh” cell products, making the  release of the product based on preliminary results from microbiological testing a necessity.For flow cytometry, not only the analysis of identity and cell counts plays an important role but also the data management as well as the reliable calculation of the required parameters.Here, we give insights to our experiences during establishment and routine performance of quality control.

About Regina Grey
Dr. Regina Gary is postdoctoral researcher working on T cell therapy at Universitätsklinikum Erlangen (UKER). Since 2018/2019, she holds the position as Head of Quality Control and Qualified Person in the Cell Processing Unit at UKER for the manufacturing of CAR T cells. From 2011–2018, she was responsible for the optimization of  manufacturing process of clinical-grade virus-specific T cells as well as the set-up and validation of quality control, and organized the manufacturing of these ATMPs after approval of the clinical trial phase I/II (EudraCT Number 2012-004240-30).  
She obtained her Ph.D. from the University of Regensburg, Germany, where she also completed her diploma studies in Biology. 

The role of flow cytometry in CAR T cell treatment

Speaker: Thomas Matthes
University of Geneva, Switzerland 

Abstract
Chimeric antigen receptor (CAR) T cells represent a highly promising new approach to treat hematological malignancies such as leukemias and lymphomas. Two CAR T cell therapies, tisagenlecleucel and axicabtagene ciloleucel, have been recently approved by FDA and EMA, and others are currently following. Flow cytometry is a powerful tool for the comprehensive phenotyping of individual CAR T cells at multiple stages of treatment, from product characterization during manufacturing and prior to infusion to longitudinal evaluation and follow-up of the infused CAR T cells in patients. The talk will focus on describing key considerations for the development, optimization, validation, and implementation of flow cytometric methods in clinical trial design and for routine monitoring of CAR T cell therapies.

About Thomas Matthes
Thomas Matthes is Associate Professor at the Hematology Service of the University of Geneva, Switzerland. He studied medicine at the Universities of Ulm (Germany), Lausanne and Geneva. After obtaining his doctorate, he spent three years at the Pasteur Institute in Paris, before specializing in Internal Medicine and later in Hematology. Since 2003 he holds a senior position (Médecin Adjoint) at the Hematology Service, and is responsible for the Diagnostics Flow Cytometry Laboratory, and since 2014 for the Diagnostics Unit. 
Dr. Matthes’ scientific work focuses on the role of adhesion molecules in the pathophysiology of lymphoproliferative syndromes and on the development of new biomarkers for the diagnosis of these diseases. He also studies the bone marrow microenvironment and hematopoietic stem cells, in particular from patients with acute myeloid leukemia, using phenotypic approaches and 3D in vitro culture systems.  
From 2010 to 2013 he was President of the Swiss Flow Cytomery Society and in 2012 he founded the Swiss Flow Cytometry School and is its current director (www.cytometryschool.ch). 

Panel discussion with Bechara Mfarrej

Institut Paoli Calmettes, Marseille, France

A translational researcher by experience, Bechara Mfarrej puts forward a multinational experience, developing laboratory- and clinical-grade ATMP for indications in solid organ transplantation and cancer for clinical studies. He obtained his Ph.D. from Utrecht University (NL) in 2019. Leading an R&D team, he delivered projects focused on automation: the devices are currently in routine use in manipulating hematopoietic progenitor grafts, NK cells, and mononuclear cell starting material. He has initiated and maintained stable collaborations with key industrial and academic stakeholders in the field of cell and gene therapy.  

ATMP and quality control: needs for continuous implementation of the analytical tools 

Speaker: Rossana Bugianesi
Pediatric Hospital Bambino Gesù, Rome, Italy

Abstract
The production of medicinal products for advanced therapies (ATMP) is a rapidly evolving cutting-edge sector. Due to the drive for innovation that characterizes the ATMP, quality control is often challenged by the needs to fully meet the GMP requirements and is committed in continuous improvement of the analytical tools.At the production site for Gene and Cellular Therapy of the Pediatric Hospital Bambino Gesù we have recently implemented a new CAR T production process by using the automated cell processing platform CliniMACS Prodigy^®.The flow cytometer MACSQuant^® Analyzer was acquired for this project and was tailored to meet specific product quality control needs. Indeed, this instrument is equipped with an algorithm-based express mode that enables automatic gating to support unbiased data analysis. Additionally, the MACSQuant Analyzer provides an automatic cells absolute count per volume that delivers highly accurate results.A further aspect related to the quality control of the product CAR T concerns the clinical requirement for a fresh product to administer within 24 hours post-production. In order to assess the microbiological safety of the product in time for the infusion the bioMérieux BacT/Alert^® 3D system was implemented. The BacT/Alert^® provides an automated microbial detection system that allows faster microbiological analysis from 14 days down to a maximum of one week. For this project, the microbiological analysis is performed in advance on an in-process control sample collected one week before the end of process. A risk assessment has shown that this approach is acceptable because the entire manufacturing process is mainly carried out in a closed system. With the aim of definitively guaranteeing the sterility of the product upon infusion, we are developing a qPCR method for the rapid microbiological detection in just 4 hours.

About Rossana Bugianesi 
Dr. Rossana Bugianesi joined the Pediatric Hospital Bambino Gesù in Rome where she currently works as Quality Control Manager for Advanced Therapy. She develops strategies for quality guarantee and verification of the processes and materials involved in production and release of advanced therapies products. Before joining the Bambino Gesù Hospital, she spent over 16 years in pharmaceutical industry covering managing roles as Department Head of Pharmacokinetics and Drug Metabolism and Director of the Clinical Laboratory. During that time, she also matured a strong know-how in analytical biochemistry. Dr. Bugianesi holds a degree in Chemistry, a Ph.D. in Experimental Physiopathology and a second level Specializing Master as Quality Systems Expert. 

Flow cytometry as analytic tool in development and quality control of cell therapy products

Speaker: Ulf Geumann
Minaris Regenerative Medicine GmbH, Ottobrunn, Germany 

Abstract
Cell-based therapies have started to offer treatment options for previously incurable diseases. A few of them have already reached marketing authorization in various areas reaching from CAR T cells against lymphoma and leukaemia, limbal stem cells for injured eyes to modified human stem cells for severe combined immunodeficiency and infusion-dependent beta thalassemia. Compared with small molecules or antibodies these genetically modified cells pose novel challenges not only regarding their manufacturing but also their analytics. Minor differences in the cellular product can decide between success and failure in the clinic. Flow cytometry offers the opportunity to analyse various parameters in parallel with single-cell resolution for an in-depth characterisation. On certain devices the volumetric, i.e. bead-free, determination of a reliable cell count has become possible, making an additional cell count process unnecessary. Even though it is a very powerful method it is not without pitfalls. Initially focussed on academia, GMP compliance aspects such as data integrity have long been neglected by the manufactures of flow cytometry devices and require particular attention. As one of the pioneers in the European ATMP field, we had to make efforts to integrate flow cytometry in the GMP environment at Minaris Regenerative Medicine GmbH (Minaris EU). In addition, due to the complexity of the device, system performance must be monitored closely to ensure consistency over time. At Minaris EU, this is done in a three-step procedure using calibration beads, Rainbow beads and Miltenyi Biotec’s 8-color immunophenotyping kit thereby spotting even minor changes in system performance and ensuring reproducibility.  

About Ulf Geumann
Dr. Ulf Geumann studied Biochemistry at the Universities of Leipzig and Glasgow. In 2009, he received his Ph.D. for work on early endosomal SNARE-proteins conducted at the Max-Planck-Institute for Biophysical Chemistry. This was followed by postdoctoral research at the Netherlands Cancer Institute on mechanisms of T cell activation. Afterwards he joined the German consortium for clinical cancer research at its site at the Klinikum rechts der Isar, Munich, applying transposon and CRISPR/Cas9 screening as well as next generation sequencing approaches. In 2016, he joined Minaris Regenerative Medicine (formerly apceth Biopharma) GmbH as a scientist and later on group leader in the preclinical development division. Since 2019, his work focuses on analytical procedures, data integrity, computerised system validation, and digitalisation under good manufacturing practise (GMP) conditions. He is currently Head of the Analytical Technology group at Minaris EU.     

Panel discussion with Dirk Windgassen

Director, Miltenyi Biotec

Dirk Windgassen, Ph.D., has 15 years of process and assay development experience in molecular diagnostics and cell therapy. He worked at Exact Sciences and Thermo Fisher Scientific where he led teams supporting the commercialization of novel molecular diagnostics assays. Prior to that he worked at Dendreon in the process development group supporting the BLA submission 

for Provenge, a first in-class cellular immunotherapy for the treatment of prostate cancer. Dirk Windgassen completed his Ph.D. thesis work on ex vivo T cell expansion at Northwestern University, USA, and graduated from the University of Erlangen-Nuremberg, Germany, in Bioprocess Engineering. 

Panel discussion with Saul Priceman

City of Hope, Duarte, USA

Saul Priceman, Ph.D., is Assistant Professor at the Departments of Hematology / Hematopoietic Cell Transplantation and Immuno-Oncology, and Associate Director of Translational Sciences in the T Cell Therapeutics Research Laboratories at City of Hope, Duarte, USA. As a principal investigator, his laboratory focuses on developing novel immunotherapy approaches for solid tumors, with

preclinical and clinical programs evaluating adoptive T cell therapies. Prior to City of Hope, Dr. Priceman received his Ph.D. in 2010 from UCLA’s department of molecular and medical pharmacology. As a postdoctoral fellow at City of Hope, his focus was on elucidating molecular mechanisms underlying the intersection between type-2 diabetes and immune-mediated breast cancer metastasis. Joining the faculty at City of Hope in 2013, the Priceman lab was charged with developing chimeric antigen receptor (CAR) T cell therapies for solid tumors, initially focusing on prostate cancer, and then on breast cancer brain metastasis and ovarian cancer. In the past 7 years, his laboratory has developed two of these programs from inception to phase 1 clinical trials, with CAR T cell therapies being investigated in the treatment of patients with metastatic castration resistant prostate cancer and brain metastases from breast cancer. Recent efforts have been made to develop similar approaches for treating advanced ovarian and pancreatic cancers, with translational programs that are anticipated to reach patients in 2021. The Priceman lab also uncovered a novel immunotherapy platform combination utilizing oncolytic viruses and CAR T cells to broadly target solid tumors, and continues to make advancements in the field related to the knowledge of immunosuppressive tumor microenvironments and combination approaches to improve therapy outcomes. 

Dewitt Jones

Regional Manager – Instrument Sales, Miltenyi Biotec

After graduating from The Citadel with a B.S. in Biology in 2010 and broadening his knowledge in the laboratory as research assistant, Dewitt Jones joined Miltenyi Biotec in 2011. Over the last decade, he has held various positions supporting the flow cytometry portfolio of Miltenyi Biotec including the roles of flow cytometry specialist, global product manager, group leader and his current role as regional manager-instruments for western North America. 

Christine Ahlert

Application Specialist, Miltenyi Biotec

Christine Ahlert joined Miltenyi Biotec as Application Specialist in 2019. She finished her master's program in Biomedical Engineering at the University of Applied Sciences in Münster, Germany, where she also received her bachelor's degree in Engineering Physics. 

Speaker: Heike Volkmer

VBC Team GmbH, Hannover, Germany

Abstract
Dr. Heike Volkmer will talk about the regulatory requirements and expectations regarding new analytical technologies and how these can be addressed in practice throughout research and development of new cell therapy products. Her presentation will outline the advantages of incorporating ICH, FDA and EMA guidelines early on in the analytical development and how to optimize the analytical transfer during product and process development from design up to commercialization. Her talk will highlight the advantages of NOT having a check-list during analytical development and qualification and emphasize the importance of DoE concepts during analytical development and validation and its advantages with regard to overall costs and timelines.

About Heike Volkmer
Dr. Heike Volkmer is a qualified biologist and endocrinologist and has almost 30 years of experience in national and international regulatory affairs, with specialist expertise in the European centralized procedure for biological, biotechnologically-derived and advanced therapy medicinal products. During this time, she has contributed to the successful approval of several innovative products requiring novel approaches during development and often special considerations during their approval processes. Over the years, Dr. Heike Volkmer has established a large number of personal contacts with representatives of the national EU authorities, the EMEA, CDER, CBER, TGA, and various other international agencies. She is a recognized expert for regulatory strategy and has authored or co-authored many CMC Expert Reports (Quality Overall Summaries). Since October 2000, Dr. Heike Volkmer is working together with her long-standing colleagues as Managing Director and Principal Consultant of the VBC Team. 

Q&A with Bernd Schröder

Global Head of Regulatory Affairs, Miltenyi Biotec

Bernd Schröder is Global Head of Regulatory Affairs at Miltenyi Biotec. He is responsible for biological and cell therapeutic medicinal products, manufacturing license, and regulatory support of lentiviral vector amongst others. Prior to joining Miltenyi Biotec, he held the position as Head of Production Fermentation, Head of Production according to §15 German Drug Law at MainGen Biotechnologie GmbH. Since 2009, Bernd Schröder is Qualified Person according to §14 German Drug Law for procurement and marketing of tissue products as well as for biopharmaceutical products since 2011.

From tissue clearing to cleared immunological processes

Speaker: Anika Grüneboom
Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany 

Abstract
Fluorescence microscopic analyses of large biological samples are subject to technical limitations due to their opacity, which causes light absorption, reflection, and scattering. These physical characteristics are particularly challenging for analyses of anatomy and physiology. However, new technology that overcomes these limitations is light sheet fluorescence microscopy, which in combination with optical tissue clearing enables three-dimensional analyses of large tissue samples. By using fluorescently labeled antibodies and obtaining endogenous fluorescence proteins, optical clearing methods together with light sheet fluorescence microscopy enable the specific identification of biological structures down to individual cells. 
According to this, we were able to extend standard techniques of clinical diagnostics with new, more specific analyses in the context of kidney diseases. Light sheet fluorescence microscopy can also be used to analyze the therapeutic efficiency of new drugs, in this case in connection with the therapeutic treatment of inflammatory bowel disease (IBD). In addition to these medically focused applications, light sheet fluorescence microscopy also represents a promising technology in basic research. For example, we were able to identify and characterize a previously unexplained blood vessel system in long bones for the first time, which makes an essential contribution to the anatomical and physiological understanding of this organ. We were also able to identify a previously unknown macrophage population in knee joints that forms a physical barrier to the joint and shields it from inflammatory immune cells. This discovery opens up completely new therapeutic approaches for joint-associated diseases such as rheumatoid arthritis or gout. These examples illustrate the wide range of applications of this technology and its high potential for future studies in the field of biology, translational research, and clinical application.

About Anika Grüneboom
Anika Grüneboom is professor at the University Hospital Essen, University Duisburg-Essen and group leader at the Leibniz-Institut für analytische Wissenschaften – ISAS – e.V., Dortmund, Germany. Prior to her current position, she worked as postdoctoral researcher and junior research group leader at the Department of Medicine 3 – Rheumatology and Immunology, Universitätsklinikum Erlangen (UKER), Germany. In 2017, Prof. Anika Grüneboom obtained her Ph.D. from the University Duisburg-Essen. She studied Biology at the RWTH Aachen, Germany. 

Unbiased and scalable AI-based imaging methods for the next-generation biomedical solutions

Speaker: Ali Ertürk
Institute of Tissue Engineering and Regnerative Medicine (iTERM), Helmholtz Zentrum München, Germany

Abstract
Ertürk lab develops and implements technologies to speed up biomedical research. In particular, we focus on new imaging approaches to visualize intact biological specimens (such as whole mouse organs and bodies, entire human organs, organoids, and engineered tissues) at the cellular and molecular level without sectioning. This enables among others to visualize cancer metastasis, infections, inflammation, neurodegeneration, and drug targeting down to singe cells in intact mice. We combined whole sample imaging with artificial intelligence and engineered tissues of human diseases for personalized drug development.

About Ali Ertürk 
Dr. Ali Ertürk graduated from Bilkent University in Ankara in 2003 and moved to Max Planck Institute for Neurobiology in Munich, Germany, for his Ph.D. where he already started working on imaging and tissue clearing in 2007. In 2009, he joined Genentech in San Francisco, USA, as a postdoc. Ali Ertürk worked on non-apoptotic caspase-3 actions in spinal degeneration, traumatic brain injury, and whole-brain tissue clearing to assess neurodegeneration. In 2014, he went back to Germany to join the Institute for Stroke and Dementia (ISD) at LMU Munich as a group leader and became a director at Helmholtz Zentrum München in 2019. In his lab, he is focusing on bringing clearing technologies to whole mouse bodies and human organs. Another focus is using deep learning in large data analysis. His group recently published several AI-based imaging methods to analyze intact biological specimens at the cell level. 

Panel discussion with Selina Keppler

Transla TUM – Center for Translational Cancer Research, Munich, Germany 

Selina Keppler did her Ph.D. in Immunology, investigating the role of cytokines in T cell activation and memory formation during infection. Continuing her research on actin regulators and their role in B cell receptor signaling, Selina Keppler spent several years as postdoc at the Francis Crick Institute London (former Cancer Research UK) in the lab of Facundo D. Batista. During her work in 

London, she first started imaging (confocal, 2-photon and TIRFM) and since then is hooked on colorful images. In late 2017, she started as Junior PI at the Center for Translational Cancer Research (TranslaTUM) in Munich. Selina Keppler and her team investigate the modulation of immune responses through the actin cytoskeleton during inflammation. In order to better understand the interplay between tissue niches, metabolism and immune cells, the Keppler lab recently implemented 3D imaging of organs or large tissue pieces in their workflow (Hofmann et al. (2000) Frontiers in Immunology 11: 314). 

Panel discussion with Bettina Weigelin

Werner Siemens Imaging Center, Tübingen, Germany

Bettina Weigelin is professor at the Werner Siemens Imaging Center in the department for Preclinical Imaging and Radiopharmacy, University of Tübingen, Germany. In 2015, she obtained her Ph.D. in Medical Sciences from the Radboud University of Nijmegen, The Netherlands, where she applied intravital multiphoton imaging to study cancer invasion and immune function in solid 

tumors. With the support of a Rubicon Young Investigator Award (NWO), she spent three years as junior faculty (Instructor) at MD Anderson Cancer Center, Houston, USA, where she used intravital microscopy to understand immunosuppression mediated by the bone microenvironment to develop novel strategies for immunotargeting of prostate cancer bone metastasis. Her current research at the University of Tübingen combines dynamic intravital microscopy with macroscopic PET/MR imaging to provide mechanistic insights into cellular therapies at the tissue and whole-body scale and to identify strategies for improved cancer immunotherapies.