Mitigation of supply chain challenges in cell therapy manufacturing: perspectives from the cord blood alliance.

Cellular therapies rely on highly specialized supply chains that often depend on single source providers. Public cord blood banks (CBB) manufacturing the first cell therapy to be highly regulated by the FDA and related international agencies are a prime example of being subject to this phenomenon. In addition to banking unrelated donor cord blood units for transplantation, CBBs also source and characterize starting materials for supply to allogeneic cell therapy developers that often employ customized technologies offered by just a small number of manufacturers. As such, these supply chains are especially sensitive to even minor changes which often result in potential major impacts. Regulations can shape supply chain efficiencies, both directly via the definition of restricted technology and process requirements and indirectly by steering strategic business decisions of critical supply or service providers. We present 3 current supply chain issues with different root causes that are swaying efficiencies in cord blood banking and beyond. Specifically, the shortage of Hespan, a common supplement used in cord blood processing, the decision by the provider to stop supporting medical device marking of the Sepax system broadly used in cord blood banking, and a new European ruling on phasing out plasticizers that are critical for providing flexibility to cord blood collection bags, are all threatening downstream supply chain issues for the biologics field. We discuss overcoming these hurdles through the prism of unified mitigation strategies, defined, and implemented by multi-factorial teams and stakeholders, to negotiate resolutions with providers and regulators alike.

Lentiviral vector determinants of anion-exchange chromatography elution heterogeneity.

The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatography remains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G]) did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1).

Current status and future prospective of breast cancer immunotherapy.

The immune system is complicated, interconnected, and offers a powerful defense system that protects its host from foreign pathogens. Immunotherapy involves boosting the immune system to kill cancer cells, and nowadays, is a major emerging treatment for cancer. With the advances in our understanding of the immunology of cancer, there has been an explosion of studies to develop and evaluate therapies that engage the immune system in the fight against cancer. Nevertheless, conventional therapies have been effective in reducing tumor burden and prolonging patient life, but the overall efficacy of these treatment regimens has been somewhat mixed and often with severe side effects. A common reason for this is the activation of molecular mechanisms that lead to apoptosis of anti-tumor effector cells. The competency to block tumor escape entirely depends on our understanding of the cellular and molecular pathways which operate in the tumor microenvironment. Numerous strategies have been developed for activating the immune system to kill tumor cells. Breast cancer is one of the major causes of cancer death in women, and is characterized by complex molecular and cellular events that closely intertwine with the host immune system. In this regard, predictive biomarkers of immunotherapy, use of nanotechnology, personalized cancer vaccines, antibodies to checkpoint inhibitors, engineered chimeric antigen receptor-T cells, and the combination with other therapeutic modalities have transformed cancer therapy and optimized the therapeutic effect. In this chapter, we will offer a holistic view of the different therapeutic modalities and recent advances in immunotherapy. Additionally, we will summarize the recent advances and future prospective of breast cancer immunotherapies, as a case study.

Development of novel lipoplex formulation methodologies to improve large-scale transient transfection for lentiviral vector manufacture.

Large-scale transient transfection has advanced significantly over the last 20 years, enabling the effective production of a diverse range of biopharmaceutical products, including viral vectors. However, a number of challenges specifically related to transfection reagent stability and transfection complex preparation times remain. New developments and improved transfection technologies are required to ensure that transient gene expression-based bioprocesses can meet the growing demand for viral vectors. In this paper, we demonstrate that the growth of cationic lipid-based liposomes, an essential step in many cationic lipid-based transfection processes, can be controlled through adoption of low pH (pH 6.40 to pH 6.75) and in low salt concentration (0.2× PBS) formulations, facilitating improved control over the nanoparticle growth kinetics and enhancing particle stability. Such complexes retain the ability to facilitate efficient transfection for prolonged periods compared with standard preparation methodologies. These findings have significant industrial applications for the large-scale manufacture of lentiviral vectors for two principal reasons. First, the alternative preparation strategy enables longer liposome incubation times to be used, facilitating effective control in a good manufacturing practices setting. Second, the improvement in particle stability facilitates the setting of wider process operating ranges, which will significantly improve process robustness and maximise batch-to-batch control and product consistency.

The highest-cost Medicaid enrollees with sickle cell disease had annual health care expenditures nearing $200 000.

Sickle cell disease (SCD) is a painful chronic blood disorder that causes serious complications and comorbidities, often leading to premature death. SCD impacts millions of people worldwide, including an estimated 100 000 in the United States, most of whom are Black or Latino. We analyzed Medicaid enrollment, claims, and encounter data via the Transformed Medicaid Statistical Information System (T-MSIS) to examine the 2021 health care utilization and spending of Medicaid enrollees with SCD. Our analysis found that Medicaid enrollees with SCD have high annual medical and pharmacy expenditures that are not evenly distributed across the population. Among the most severe enrollees with genotypes eligible for clinical trials, those in the top 5% of health care spending incurred, on average, nearly $200 000 per year for this chronic condition.

Polish regulatory system regarding ATMP hospital exemptions.

Introduction: This article explains the current regulatory system in Poland regarding Advanced Therapy Medicinal Products given under Hospital Exemptions (ATMP-HE). Methods: The relevant sections of Polish legislation are translated into English and their interaction is described. Results: We analyze the impact of these regulations from the perspective of three stakeholder groups: manufacturers, physicians, and patients. Amendments enacted between 2018 and 2023 have substantially changed Polish implementation of the ATMP-HE pathway. In Poland, most ATMP-HE treatments have been therapies employing Mesenchymal Stromal Cells (MSC). Discussion: Comparison to other European countries shows that Poland is within the mainstream of EU practices regarding ATMP-HE implementation. One notable issue is that Poland has relatively low per capita spending on healthcare, and ATMP-HE in Poland must be funded from outside the government healthcare system. Conclusions. The original intention of the legislation that created ATMP-HE was to allow access to experimental therapies for patients with unmet needs. It remains to be seen if that mission can be fulfilled amidst conflicting pressures from various stakeholder groups.

Multiple treatment interruptions and protecting HIV-specific CD4 T cells enable durable CD8 T cell response and viral control.

Human Immunodeficiency Virus (HIV) remains a global health challenge, and novel approaches to improve HIV control are significantly important. The cell and gene therapy product AGT103-T was previously evaluated (NCT04561258) for safety, immunogenicity, and persistence in seven patients for up to 180 days post infusion. In this study, we sought to investigate the impact of AGT103-T treatment upon analytical treatment interruptions (ATIs). Six patients previously infused with AGT103-T were enrolled into an ATI study (NCT05540964), wherein they suspended their antiretroviral therapy (ART) until their viral load reached 100,000 copies/mL in two successive visits, or their CD4 count was reduced to below 300 cells/µL. During the ATI, all patients experienced viral rebound followed by a notable expansion in HIV specific immune responses. The participants demonstrated up to a five-fold increase in total CD8 counts over baseline approximately 1-2 weeks followed by the peak viremia. This coincided with a rise in HIV-specific CD8 T cells, which was attributed to the increase in antigen availability and memory recall. Thus, the protocol was amended to include a second ATI with the first ATI serving as an "auto-vaccination." Four patients participated in a second ATI. During the second ATI, the Gag-specific CD8 T cells were either maintained or rose in response to viral rebound and the peak viremia was substantially decreased. The patients reached a viral set point ranging from 7,000 copies/mL to 25,000 copies/mL. Upon resuming ART, all participants achieved viral control more rapidly than during the first ATI, with CD4 counts remaining within 10% of baseline measurements and without any serious adverse events or evidence of drug resistance. In summary, the rise in CD8 counts and the viral suppression observed in 100% of the study participants are novel observations demonstrating that AGT103-T gene therapy when combined with multiple ATIs, is a safe and effective approach for achieving viral control, with viral setpoints consistently below 25,000 copies/mL and relatively stable CD4 T cell counts. We conclude that HIV cure-oriented cell and gene therapy trials should include ATI and may benefit from designs that include multiple ATIs when induction of CD8 T cells is required to establish viral control.

Challenges and Lessons Learned in Autologous Chimeric Antigen Receptor T-Cell Therapy Development from a Statistical Perspective.

Chimeric antigen receptor (CAR) T-cell therapy is a human gene therapy product where T cells from a patient are genetically modified to enable them to recognize desired target antigen(s) more effectively. In recent years, promising antitumor activity has been seen with autologous CAR T cells. Since 2017, six CAR T-cell therapies for the treatment of hematological malignancies have been approved by the Food and Drug Administration (FDA). Despite the rapid progress of CAR T-cell therapies, considerable statistical challenges still exist for this category of products across all phases of clinical development that need to be addressed. These include (but not limited to) dose finding strategy, implementation of the estimand framework, use of real-world data in contextualizing single-arm CAR T trials, analysis of safety data and long-term follow-up studies. This paper is the first step in summarizing and addressing these statistical hurdles based on the development of the six approved CAR T-cell products.

Assessing biologic/toxicologic effects of extractables from plastic contact materials for advanced therapy manufacturing using cell painting assay and cytotoxicity screening.

Plastic components are essential in the pharmaceutical industry, encompassing container closure systems, laboratory handling equipment, and single-use systems. As part of their material qualification process, studies on interactions between plastic contact materials and process solutions or drug products are conducted. The assessment of single-use systems includes their potential impact on patient safety, product quality, and process performance. This is particularly crucial in cell and gene therapy applications since interactions with the plastic contact material may result in an adverse effect on the isolated therapeutic human cells. We utilized the cell painting assay (CPA), a non-targeted method, for profiling the morphological characteristics of U2OS human osteosarcoma cells in contact with chemicals related to plastic contact materials. Specifically, we conducted a comprehensive analysis of 45 common plastic extractables, and two extracts from single-use systems. Results of the CPA are compared with a standard cytotoxicity assay, an osteogenesis differentiation assay, and in silico toxicity predictions. The findings of this feasibility study demonstrate that the device extracts and most of the tested compounds do not evoke any measurable biological changes on the cells (induction ≤ 5%) among the 579 cell features measured at concentrations ≤ 50 µM. CPA can serve as an important assay to reveal unique information not accessible through quantitative structure-activity relationship analysis and vice versa. The results highlight the need for a combination of in vitro and in silico methods in a comprehensive assessment of single-use equipment utilized in advanced therapy medicinal products manufacturing.

Performance and Stability of New Class of Fetal Bovine Sera (FBS) and Its Lyophilized Form in ELISpot and FluoroSpot Assays: Applications for Monitoring the Immune Response in Vaccine, and Cell and Gene Immunotherapy in Clinical Trials.

Interferon-gamma (IFNγ) ELISpot and FluoroSpot are widely used assays to detect functional cell responses in immunotherapy clinical studies. Recognized for their importance in vaccine development studies to quantitate immune responses, these assays have more recently risen to the forefront in cell and gene therapy as well as cancer immunotherapy fields where responses against cancer neoantigens are not easily detectable above assay background. Here, we test a new class of fetal bovine serum (FBS), CultraPure FBS, in ex vivo ELISpot and FluoroSpot assays and cultured FluoroSpot assays following in vitro expansion. Several CultraPure FBS lots that have been specially formulated through the process of lyophilization (lyo-FBS) were compared to liquid CultraPure FBS. We stimulated human PBMCs with antigen-specific peptide pools diluted in media supplemented with liquid CultraPure FBS or lyo-FBS and found equivalent cytokine production with negligible to no assay background with both liquid and lyo-FBS formats. Moreover, the lyo-FBS showed lot-to-lot consistency and 90-day refrigerated (4 °C) stability in both ex vivo direct and in vitro cultured assays. In addition, we present here a method using lyo-FBS for the expansion of low-frequency antigen-specific T cells, mimicking the low frequency seen with cancer neoantigens by utilizing a cultured FluoroSpot assay. Our results demonstrate the presence of Granzyme B, interferon-gamma (IFNγ), and tumor necrosis factor (TNF) production by antigen-specific polyfunctional T cells following a 9-day culture using media supplemented with lyo-FBS.

Cell and gene therapy investment: evolution and future outlook on investor perspectives.

BACKGROUND AIMS: To better understand the attitudes and behaviors of investors involved in funding cell and gene therapy (CGT) businesses, the Business Development and Finance) subcommittee of International Society for Cell and Gene Therapy, in collaboration with Truist Securities, conducted a broad survey of the investment community in late 2021. METHODS: This survey follows a similar study that this group executed in 2018, and the longitudinal comparisons between the two time periods provide insights into how investor behavior in the CGT field has evolved. RESULTS: The vast majority of investor respondents are specialist biotech investors who are primarily active in deploying capital in North America and Europe. There was a notable increase in the proportion of investors actively deploying capital in China and Japan between 2018 and 2021. The percentage of respondents' portfolios dedicated to CGT companies has also increased in this period, reflecting a noteworthy trend in the therapeutic landscape. CONCLUSIONS: Clinically significant data remain the dominant force behind investment decisions, whereas competition from other drug modalities has now emerged as the most-cited barrier to making a CGT investment, eclipsing safety concerns as the most significant barrier to investment in 2018. Concerns around manufacturing and scale-up have also increased in prominence amongst the investment community. Gene-editing technologies are attracting investors as the most compelling new CGT technology. This survey also revealed that most investors expect to increase their level of investment in allogeneic technologies relative to autologous products in the coming years.

Real-world evidence for coverage determination of treatments for rare diseases.

Health technology assessment (HTA) decisions for pharmaceuticals are complex and evolving. New rare disease treatments are often approved more quickly through accelerated approval schemes, creating more uncertainties about clinical evidence and budget impact at the time of market entry. The use of real-world evidence (RWE), including early coverage with evidence development, has been suggested as a means to support HTA decisions for rare disease treatments. However, the collection and use of RWE poses substantial challenges. These challenges are compounded when considered in the context of treatments for rare diseases. In this paper, we describe the methodological challenges to developing and using prospective and retrospective RWE for HTA decisions, for rare diseases in particular. We focus attention on key elements of study design and analyses, including patient selection and recruitment, appropriate adjustment for confounding and other sources of bias, outcome selection, and data quality monitoring. We conclude by offering suggestions to help address some of the most vexing challenges. The role of RWE in coverage and pricing determination will grow. It is, therefore, necessary for researchers, manufacturers, HTA agencies, and payers to ensure that rigorous and appropriate scientific principles are followed when using RWE as part of decision-making.

Recent advances in CRISPR-Cas9-based genome insertion technologies.

Programmable genome insertion (or knock-in) is vital for both fundamental and translational research. The continuously expanding number of CRISPR-based genome insertion strategies demonstrates the ongoing development in this field. Common methods for site-specific genome insertion rely on cellular double-strand breaks repair pathways, such as homology-directed repair, non-homologous end-joining, and microhomology-mediated end joining. Recent advancements have further expanded the toolbox of programmable genome insertion techniques, including prime editing, integrase coupled with programmable nuclease, and CRISPR-associated transposon. These tools possess their own capabilities and limitations, promoting tremendous efforts to enhance editing efficiency, broaden targeting scope and improve editing specificity. In this review, we first summarize recent advances in programmable genome insertion techniques. We then elaborate on the cons and pros of each technique to assist researchers in making informed choices when using these tools. Finally, we identify opportunities for future improvements and applications in basic research and therapeutics.

Current challenges in cell and gene therapy: a joint view from the European Committee of the International Society for Cell & Gene Therapy (ISCT) and the European Society for Blood and Marrow Transplantation (EBMT).

Cell and gene therapy poses evolving challenges. The current article summarizes the discussions held by European Regional Committee of the International Society for Cell & Gene Therapy and the European Society for Blood and Marrow Transplantation (EBMT) on the current challenges in this field, focusing on the European setting. This article emphasizes the imperative assessment of real-world cell and gene therapy activity, advocating for expanded registries beyond hematopoietic transplantation and chimeric antigen receptor-T-cell therapy. Accreditation's role in ensuring standardized procedures, as exemplified by JACIE (The Joint Accreditation Committee of ISCT-Europe and EBMT), is crucial for safety. Access to commercial products and reimbursement variations among countries underscore the need for uniform access to advanced therapy medical products (ATMPs). Academic product development and point-of-care manufacturing face barriers to patient access. Hospital Exemption's potential, demonstrated by some initial experiences, may increase patient accessibility in individual situations. Regulatory challenges, including the ongoing European ATMPs legislation review, necessitate standardized criteria for Hospital Exemption and mandatory reporting within registries. Efforts to combat unproven therapies and fraud involve collaboration between scientific societies, regulatory bodies and patient groups. Finally, is important to highlight the vital role of education and workforce development in meeting the escalating demand for specialized professionals in the ATMP field. Collaboration among scientific societies, academic institutions, industry, regulatory bodies and patient groups is crucial for overcoming all these challenges to increase gene and cell therapy activity in Europe.

Cell and Gene Therapies: Challenges in Designing Extractables and Leachables Studies and Conducting Safety Assessments.

Over the past decade, Cell and Gene Therapies (C>) have been an emerging therapeutic area with more than twenty C> drug products approved and over 1000 registered trials. The remarkable progress in these modalities brings new challenges for scientists who evaluate manufacturing and storage materials, including risk assessments for extractables and leachables (E&L). Establishing a business process to qualify materials for these applications is an important risk mitigation strategy in support of these assessments. Process validation verifying process performance and product quality requirements using qualified materials also ensures that leachables from the materials do not result in an impact to process and product. The authors provide an overview of available guidelines and publications relevant to E&L risk assessments that can be used to support ex vivo C> products, highlighting gaps and standardization needs in the areas of biocompatibility and extractables conditions. Finally, the authors present leachable testing strategies, relevant to the specific manufacturing and storage conditions of C> products, and safety assessment considerations for organic and inorganic chemical entities.

Redefining informed consent form in cell and gene therapy trials.

Informed consent is a foundation of the ethical conduct of research involving human participants. Based on the ethical principle of respect for persons, the goal of informed consent is to ensure that participants are aware of the risks and potential benefits and make a voluntary decision about participating in clinical trial research. The extraordinary scientific advances happening globally, have demonstrated the potential of regenerative therapies in transforming the health of the nation by providing a therapeutic option for diseases that were previously considered incurable. These therapies, which include cells and gene therapy (GT) labeled as Advanced Therapeutic Medicinal Products globally, have complex mechanisms of action. Owing to their highly personalized and intricate nature of these therapies, developing the latter often presents unique challenges above and beyond those encountered for small molecule drugs. We recently looked through some cell and GT clinical trials and realized the lacunae in the informed consent form (ICF) provided by the investigators. Especially in a country like India, where the general understanding and perception of patients is limited regarding clinical trials, it is felt that any lapses in the consent process may jeopardize the informed decision-making and safety of the participants and tarnish the reputation of India globally. The present article highlights the need for appropriate patient and public education on the various aspects of cell and gene therapies and aims to address all the elements of ICF in light of the challenges associated with these innovative therapies.

Study on hospital exemption clause of advanced therapy medicinal products in EU and its enlightenment to China / 中国药房

OBJECTIVE To study the hospital exemption clause of advanced therapy medicinal products in the EU， and to provide policy recommendations for improving the regulatory system of cell and gene therapy （CGT） products in China. METHODS Through literature review and investigation of the official websites of EU member states， this study compared the differences in the application and implementation of the hospital exemption clause among member states from the perspectives of “non-conventional” definition， manufacturing standards， and pharmacovigilance requirements； the potential issues of hospital exemption clauses in practice were analyzed to propose policy recommendations based on the regulatory status of CGT in China. RESULTS & CONCLUSIONS EU has provided patients with rare diseases， who lack effective treatment or better therapy plans， with the opportunity to obtain new treatments through the hospital exemption clause， which has effectively improved the accessibility of medicines for patients. However， there still are certain disparities in the provisions of hospital exemption clause among EU member states. For instance， some member states have not explicitly defined “unconventional” circumstances； each member state has different requirements regarding production quality standards and pharmacovigilance requirement. Additionally， in the practical implementation of hospital exemption clause， issues such as poor transparency of information and a lack of certain restrictive conditions persist. Therefore， considering the current landscape and regulation of China’s CGT， it is recommended that China explore the clinical translational application of low-risk CGT in “unconventional” situations， strengthen the management of clinical translational application in terms of production quality standards and pharmacovigilance requirement. At the same time， it is necessary to further standardize the investigator initiated trials， and pay attention to the balance between clinical application and drug registration and marketing， thereby guiding the sustained and healthy development of China’s CGT.

Study on hospital exemption clause of advanced therapy medicinal products in EU and its enlightenment to China / 中国药房

OBJECTIVE To study the hospital exemption clause of advanced therapy medicinal products in the EU， and to provide policy recommendations for improving the regulatory system of cell and gene therapy （CGT） products in China. METHODS Through literature review and investigation of the official websites of EU member states， this study compared the differences in the application and implementation of the hospital exemption clause among member states from the perspectives of “non-conventional” definition， manufacturing standards， and pharmacovigilance requirements； the potential issues of hospital exemption clauses in practice were analyzed to propose policy recommendations based on the regulatory status of CGT in China. RESULTS & CONCLUSIONS EU has provided patients with rare diseases， who lack effective treatment or better therapy plans， with the opportunity to obtain new treatments through the hospital exemption clause， which has effectively improved the accessibility of medicines for patients. However， there still are certain disparities in the provisions of hospital exemption clause among EU member states. For instance， some member states have not explicitly defined “unconventional” circumstances； each member state has different requirements regarding production quality standards and pharmacovigilance requirement. Additionally， in the practical implementation of hospital exemption clause， issues such as poor transparency of information and a lack of certain restrictive conditions persist. Therefore， considering the current landscape and regulation of China’s CGT， it is recommended that China explore the clinical translational application of low-risk CGT in “unconventional” situations， strengthen the management of clinical translational application in terms of production quality standards and pharmacovigilance requirement. At the same time， it is necessary to further standardize the investigator initiated trials， and pay attention to the balance between clinical application and drug registration and marketing， thereby guiding the sustained and healthy development of China’s CGT.

Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Intact Mass Analysis of Full-Length AAV Capsid Proteins.

Adeno-associated viruses (AAVs) are commonly used as vectors for the delivery of gene therapy targets. Characterization of AAV capsid proteins (VPs) and their post-translational modifications (PTMs) have become a critical attribute monitored to evaluate product quality. Liquid chromatography-mass spectrometry (LC-MS) analysis of intact AAV VPs provides both quick and reliable serotype identification as well as proteoform information on each VP. Incorporating these analytical strategies into rapid good manufacturing practice (GMP)-compliant workflows containing robust, but simplified, data processing methods is necessary to ensure effective product quality control (QC) during production. Here, we present a GMP-compliant LC-MS workflow for the rapid identification and in-depth characterization of AAVs. Hydrophilic interaction liquid chromatography (HILIC) MS with difluoroacetic acid as a mobile phase modifier is utilized to achieve the intact separation and identification of AAV VPs and their potential proteoforms. Peptide mapping is performed to confirm PTMs identified during intact VP analysis and for in-depth PTM characterization. The intact separations platform is then incorporated into a data processing workflow developed using GMP-compliant software capable of rapid AAV serotype identification and, if desired, specific serotype PTM monitoring and characterization. Such a platform provides product QC capabilities that are easily accessible in a regulatory setting.