Upskilling the cell therapy manufacturing workforce: design, implementation, and evaluation of a massive open online course.

Cell therapies have gained prominence as a promising therapeutic modality for treating a range of diseases. Despite the recent clinical successes of cell therapy products, very few formal training programs exist for cell therapy manufacturing. To meet the demand for a well-trained workforce, we assembled a team of university researchers and industry professionals to develop an online course on the principles and practice of cell therapy manufacturing. The course covers the basic cell and systems physiology underlying cell therapy products, in addition to explaining end-to-end manufacturing from cell acquisition through to patient treatment, industrialization, and regulatory processes. So far, over 10,000 learners have enrolled in the course, and over 90% of respondents to the course exit survey indicated that they were 'very likely' or 'likely' to recommend the course to a peer. In this paper, we discuss our experience in the collaborative design and implementation of the online course, as well as lessons learned from quantitative and qualitative student feedback. We believe that this course can serve as a model for how academia and industry can collaborate to create innovative, scalable training programs to meet the demands of the modern biotechnology workforce.

Primary cutaneous marginal zone lymphoproliferative disorder following COVID-19 vaccination.

During the coronavirus disease 2019 (COVID-19) pandemic, COVID-19 vaccines were administered worldwide. A number of skin reactions, including primary cutaneous T-cell lymphoproliferative disorders (LPDs) were reported following COVID-19 vaccination. We report a case of primary cutaneous marginal zone lymphoproliferative disorder (PCMZLPD) secondary to COVID-19 vaccination. A 57-year-old man presented with an erythematous nodule on his left arm at the site of vaccine inoculation following his first dose of the Moderna (mRNA-1273) vaccine a few weeks prior. The nodule continued to progress in size after the second dose. A skin biopsy specimen of the nodule showed a diffuse dermal infiltrate of small to medium-sized lymphocytes with plasma cells and histiocytes. The infiltrate was composed of CD3+ T cells with CD20+ and CD79a+ B cells. The neoplastic B cells reacted with BCL-2 and were negative for BCL-6 and CD10. Kappa light chain restriction was identified by in situ hybridization. Gene rearrangement studies revealed kappa light chain monoclonality, confirming the diagnosis of PCMZLPD. The temporal association with the Moderna vaccination and the occurrence of the lesion at the inoculation site indicate a COVID-19 vaccination-induced PCMZLPD. This is one of the rare cases of PCMZLPD following COVID-19 vaccination.

Mechanistic modeling explains the production dynamics of recombinant adeno-associated virus with the baculovirus expression vector system.

Current manufacturing processes for recombinant adeno-associated viruses (rAAVs) have less-than-desired yields and produce significant amounts of empty capsids. The increasing demand and the high cost of goods for rAAV-based gene therapies motivate development of more efficient manufacturing processes. Recently, the US Food and Drug Administration (FDA) approved the first rAAV-based gene therapy product manufactured in the baculovirus expression vector system (BEVS), a technology that demonstrated production of high titers of full capsids. This work presents a first mechanistic model describing the key extracellular and intracellular phenomena occurring during baculovirus infection and rAAV maturation in the BEVS. The model predictions are successfully validated for in-house and literature experimental measurements of the vector genome and of structural and non-structural proteins collected during rAAV manufacturing in the BEVS with the TwoBac and ThreeBac constructs. A model-based analysis of the process is carried out to identify the bottlenecks that limit full capsid formation. Vector genome amplification is found to be the limiting step for rAAV production in Sf9 cells using either the TwoBac or ThreeBac system. In turn, vector genome amplification is hindered by limiting Rep78 levels. Transgene and non-essential baculovirus protein expression in the insect cell during rAAV manufacturing also negatively influences the rAAV production yields.

Machine learning-based detection of adventitious microbes in T-cell therapy cultures using long-read sequencing.

Assuring that cell therapy products are safe before releasing them for use in patients is critical. Currently, compendial sterility testing for bacteria and fungi can take 7-14 days. The goal of this work was to develop a rapid untargeted approach for the sensitive detection of microbial contaminants at low abundance from low volume samples during the manufacturing process of cell therapies. We developed a long-read sequencing methodology using Oxford Nanopore Technologies MinION platform with 16S and 18S amplicon sequencing to detect USP <71> organisms and other microbial species. Reads are classified metagenomically to predict the microbial species. We used an extreme gradient boosting machine learning algorithm (XGBoost) to first assess if a sample is contaminated, and second, determine whether the predicted contaminant is correctly classified or misclassified. The model was used to make a final decision on the sterility status of the input sample. An optimized experimental and bioinformatics pipeline starting from spiked species through to sequenced reads allowed for the detection of microbial samples at 10 colony-forming units (CFU)/mL using metagenomic classification. Machine learning can be coupled with long-read sequencing to detect and identify sample sterility status and microbial species present in T-cell cultures, including the USP <71> organisms to 10 CFU/mL. IMPORTANCE This research presents a novel method for rapidly and accurately detecting microbial contaminants in cell therapy products, which is essential for ensuring patient safety. Traditional testing methods are time-consuming, taking 7-14 days, while our approach can significantly reduce this time. By combining advanced long-read nanopore sequencing techniques and machine learning, we can effectively identify the presence and types of microbial contaminants at low abundance levels. This breakthrough has the potential to improve the safety and efficiency of cell therapy manufacturing, leading to better patient outcomes and a more streamlined production process.

Historical evaluation of the in vivo adventitious virus test and its potential for replacement with next generation sequencing (NGS).

The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product.

MIT CAACB Risk Assessment Case Study: Assessing Virus Cross-Contamination Risk between Two Simultaneous Processes in an Open Biomanufacturing Facility.

Some members of MIT's Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) previously published content on the "Quality Risk Management in the Context of Viral Contamination", which described tools, procedures, and methodologies for assessing and managing the risk of a potential virus contamination in cell culture processes. To address the growing industry interest in moving manufacturing toward open ballrooms with functionally closed systems and to demonstrate how the ideas of risk management can be leveraged to perform a risk assessment, CAACB conducted a case study exercise of these new manufacturing modalities. In the case study exercise, a cross-functional team composed of personnel from many of CAACB's industry membership collaboratively assessed the risks of viral cross-contamination between a human and non-human host cell system in an open manufacturing facility. This open manufacturing facility had no walls to provide architectural separation of two processes occurring simultaneously, specifically a recombinant protein perfusion cell culture process using the human cell line, HEK-293 (Process 1) and a downstream postviral filtration unit operation (Process 2) of a recombinant protein produced in CHO cells. This viral risk assessment focused on cross-contamination of the Process 2 filtration unit operation after the Process 1 perfusion bioreactor was contaminated with a virus that went undetected. The workflow for quality risk management that is recommended by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) was followed, which included identifying and mapping the manufacturing process, defining the risk question, risk evaluation, and risk control. The case study includes a completed Failure Mode and Effects Analysis (FMEA) to provide descriptions of the specific risks and corresponding recommended risk reduction actions.

The ratio of nicotinic acid to nicotinamide as a microbial biomarker for assessing cell therapy product sterility.

Controlling microbial risks in cell therapy products (CTPs) is important for product safety. Here, we identified the nicotinic acid (NA) to nicotinamide (NAM) ratio as a biomarker that detects a broad spectrum of microbial contaminants in cell cultures. We separately added six different bacterial species into mesenchymal stromal cell and T cell culture and found that NA was uniquely present in these bacteria-contaminated CTPs due to the conversion from NAM by microbial nicotinamidases, which mammals lack. In cells inoculated with 1 × 104 CFUs/mL of different microorganisms, including USP <71> defined organisms, the increase in NA to NAM ratio ranged from 72 to 15,000 times higher than the uncontaminated controls after 24 h. Importantly, only live microorganisms caused increases in this ratio. In cells inoculated with 18 CFUs/mL of Escherichia coli, 20 CFUs/mL of Bacillus subtilis, and 10 CFUs/mL of Candida albicans, significant increase of NA to NAM ratio was detected using LC-MS after 18.5, 12.5, and 24.5 h, respectively. In contrast, compendial sterility test required >24 h to detect the same amount of these three organisms. In conclusion, the NA to NAM ratio is a useful biomarker for detection of early-stage microbial contaminations in CTPs.

Weighing the DNA Content of Adeno-Associated Virus Vectors with Zeptogram Precision Using Nanomechanical Resonators.

Quantifying the composition of viral vectors used in vaccine development and gene therapy is critical for assessing their functionality. Adeno-associated virus (AAV) vectors, which are the most widely used viral vectors for in vivo gene therapy, are typically characterized using PCR, ELISA, and analytical ultracentrifugation which require laborious protocols or hours of turnaround time. Emerging methods such as charge-detection mass spectroscopy, static light scattering, and mass photometry offer turnaround times of minutes for measuring AAV mass using optical or charge properties of AAV. Here, we demonstrate an orthogonal method where suspended nanomechanical resonators (SNR) are used to directly measure both AAV mass and aggregation from a few microliters of sample within minutes. We achieve a precision near 10 zeptograms which corresponds to 1% of the genome holding capacity of the AAV capsid. Our results show the potential of our method for providing real-time quality control of viral vectors during biomanufacturing.

Large-scale cultured meat production: Trends, challenges and promising biomanufacturing technologies.

Food systems of the future will need to face an increasingly clear reality - that a protein-rich diet is essential for good health, but traditional meat products will not suffice to ensure safety, sustainability, and equity of food supply chains at a global scale. This paper provides an in-depth analysis of bioprocess technologies needed for cell-based meat production and challenges in reaching commercial scale. Specifically, it reviews state-of-the-art bioprocess technologies, current limitations, and opportunities for research across four domains: cell line development, cell culture media, scaffolding, and bioreactors. This also includes exploring innovations to make cultured meat a viable protein alternative across numerous key performance indicators and for specific applications where traditional livestock is not an option (e.g., local production, space exploration). The paper explores tradeoffs between production scale, product quality, production cost, and footprint over different time horizons. Finally, a discussion explores various factors that may impact the ability to successfully scale and market cultured meat products: social acceptance, environmental tradeoffs, regulatory guidance, and public health benefits. While the exact nature of the transition from traditional livestock to alternative protein products is uncertain, it has already started and will likely continue to build momentum in the next decade.

Rapid, robust, and sustainable antibody responses to mRNA COVID-19 vaccine in convalescent COVID-19 individuals.

Longitudinal studies are needed to evaluate the SARS-CoV-2 mRNA vaccine antibody response under real-world conditions. This longitudinal study investigated the quantity and quality of SARS-CoV-2 antibody response in 846 specimens from 350 patients, comparing BNT162b2-vaccinated individuals (19 previously diagnosed with COVID-19, termed RecoVax; and 49 never diagnosed, termed NaiveVax) with 122 hospitalized unvaccinated (HospNoVax) and 160 outpatient unvaccinated (OutPtNoVax) COVID-19 patients. NaiveVax experienced delay in generating SARS-CoV-2 total antibodies (TAb) and surrogate neutralizing antibodies (SNAb) after the first vaccine dose (D1) but rapid increase in antibody levels after the second dose (D2). However, these never reached RecoVax's robust levels. In fact, NaiveVax TAb and SNAb levels decreased 4 weeks after D2. For the most part, RecoVax TAb persisted, after reaching maximal levels 2 weeks after D2, but SNAb decreased significantly about 6 months after D1. Although NaiveVax avidity lagged behind that of RecoVax for most of the follow-up periods, NaiveVax did reach similar avidity by about 6 months after D1. These data suggest that 1 vaccine dose elicits maximal antibody response in RecoVax and may be sufficient. Also, despite decreasing levels in TAb and SNAb over time, long-term avidity may be a measure worth evaluating and possibly correlating to vaccine efficacy.

Mechanistic model for production of recombinant adeno-associated virus via triple transfection of HEK293 cells.

Manufacturing of recombinant adeno-associated virus (rAAV) viral vectors remains challenging, with low yields and low full:empty capsid ratios in the harvest. To elucidate the dynamics of recombinant viral production, we develop a mechanistic model for the synthesis of rAAV viral vectors by triple plasmid transfection based on the underlying biological processes derived from wild-type AAV. The model covers major steps starting from exogenous DNA delivery to the reaction cascade that forms viral proteins and DNA, which subsequently result in filled capsids, and the complex functions of the Rep protein as a regulator of the packaging plasmid gene expression and a catalyst for viral DNA packaging. We estimate kinetic parameters using dynamic data from literature and in-house triple transient transfection experiments. Model predictions of productivity changes as a result of the varied input plasmid ratio are benchmarked against transfection data from the literature. Sensitivity analysis suggests that (1) the poorly coordinated timeline of capsid synthesis and viral DNA replication results in a low ratio of full virions in harvest, and (2) repressive function of the Rep protein could be impeding capsid production at a later phase. The analyses from the mathematical model provide testable hypotheses for evaluation and reveal potential process bottlenecks that can be investigated.

Cellular pathways of recombinant adeno-associated virus production for gene therapy.

Recombinant adeno-associated viruses (rAAVs) are among the most important vectors for in vivo gene therapies. With the rapid development of gene therapy, current rAAV manufacturing capacity faces a challenge to meet the emerging demand for these therapies in the future. To examine the bottlenecks in rAAV production during cell culture, we focus here on an analysis of cellular pathways of rAAV production, based on an overview of assembly mechanisms first in the wild-type (wt) AAV replication and then in the common methods of rAAV production. The differences analyzed between the wild-type and recombinant systems provide insights into the mechanistic differences that may correlate with viral productivity. Based on these analyses, we identify potential barriers to high productivity of rAAV and discuss future directions for improvement to meet the emerging needs set by the growth of rAAV-based therapy and the needs of patients.

Revisiting mouse minute virus inactivation by high temperature short time treatment.

In recent years, high temperature short time (HTST) treatment technology has been increasingly adopted for medium treatment to mitigate the potential risk of viral contamination in mammalian cell culture GMP manufacturing facilities. Mouse minute virus (MMV), also called minute virus of mice (MVM), implicated in multiple viral contamination events is commonly used as a relevant model virus to assess the effectiveness of HTST treatment of cell culture media. However, results from different studies vary broadly in inactivation kinetics as well as log reduction factors (LRFs) achieved under given treatment conditions. To determine whether the reported discrepancies stemmed from differences in MMV strains, laboratory-scale HTST devices, medium matrices, and/or experimental designs, we have taken a collaborative approach to systematically assess the effectiveness of HTST treatment for MMV inactivation. This effort was conceptualized based on a media treatment gap analysis conducted by the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) under the MIT Center for Biomedical Innovation (CBI). Specifically, two different MMV strains were used to evaluate the effectiveness of HTST at various treatment conditions with regard to exposure temperature and hold time duration by two independent laboratories within two different companies. To minimize experimental variations, the two sites used the same batches of MMV stocks, the same commercially purchased medium, and the same model of thermocyclers as the laboratory-scale HTST device. The two independent laboratories yielded similar MMV inactivation kinetics and comparable LRF. No significant differences were observed between the two MMV strains evaluated, suggesting that the variations from prior studies were likely due to differences in equipment, medium matrices, or other factors. The data presented here indicate that MMV inactivation by HTST treatment obeys first-order kinetics and can be mathematically modeled using an Arrhenius equation. The model-based extrapolation provides a quantitative estimate of MMV inactivation by the current industry standard HTST condition (102°C for a hold time of 10 s) used for medium treatment. Finally, based on the data from the current study and the industry experience, it is recommended that any alternative virus barrier technologies adopted for medium treatment should provide a clearance of at least 3.0 LRF based on a worst-case model virus to effectively mitigate potential risks of viral contamination.

Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies.

The optimization of upstream and downstream processes for production of recombinant adeno-associated virus (rAAV) with consistent quality depends on the ability to rapidly characterize critical quality attributes (CQAs). In the context of rAAV production, the virus titer, capsid content, and aggregation are identified as potential CQAs, affecting the potency, purity, and safety of rAAV-mediated gene therapy products. Analytical methods to measure these attributes commonly suffer from long turnaround times or low throughput for process development, although rapid, high-throughput methods are beginning to be developed and commercialized. These methods are not yet well established in academic or industrial practice, and supportive data are scarce. Here, we review both established and upcoming analytical methods for the quantification of rAAV quality attributes. In assessing each method, we highlight the progress toward rapid, at-line characterization of rAAV. Furthermore, we identify that a key challenge for transitioning from traditional to newer methods is the scarcity of academic and industrial experience with the latter. This literature review serves as a guide for the selection of analytical methods targeting quality attributes for rapid, high-throughput process characterization during process development of rAAV-mediated gene therapies.

Convalescent plasma to treat coronavirus disease 2019 (COVID-19): considerations for clinical trial design.

Case series studying convalescent plasma use in the treatment of COVID-19 have been promising, but additional, high-quality studies are needed to determine the efficacy of the treatment when applied for prophylaxis, for early phases of illness, and for severe illness. Previous studies of convalescent plasma in treating other viral diseases have identified factors to consider when designing treatment protocols, including timing of administration relative to onset of illness, timing of donation relative to resolution of symptoms, severity of illness of the donor, pretransfusion serology of the recipient, and antibody titers of the donor. There are many clinical trials studying treatment of, and prophylaxis against, COVID-19 using convalescent plasma. In addition to clinical trials, the FDA also allows treatment through two other pathways: the "Expanded Access to Convalescent Plasma for the Treatment of Patients with COVID-19" protocol, and emergency investigational new drug applications. The FDA also provides criteria for donation of convalescent plasma.

Viral contamination in biologic manufacture and implications for emerging therapies.

Recombinant protein therapeutics, vaccines, and plasma products have a long record of safety. However, the use of cell culture to produce recombinant proteins is still susceptible to contamination with viruses. These contaminations cost millions of dollars to recover from, can lead to patients not receiving therapies, and are very rare, which makes learning from past events difficult. A consortium of biotech companies, together with the Massachusetts Institute of Technology, has convened to collect data on these events. This industry-wide study provides insights into the most common viral contaminants, the source of those contaminants, the cell lines affected, corrective actions, as well as the impact of such events. These results have implications for the safe and effective production of not just current products, but also emerging cell and gene therapies which have shown much therapeutic promise.

Biopharmaceutical Industry Approaches to Facility Segregation for Viral Safety: An Effort from the Consortium on Adventitious Agent Contamination in Biomanufacturing.

Appropriate segregation within manufacturing facilities is required by regulators and utilized by manufacturers to ensure that the final product has not been contaminated with (a) adventitious viruses, (b) another pre-/postviral clearance fraction of the same product, or (c) another product processed in the same facility. However, there is no consensus on what constitutes appropriate facility segregation to minimize these risks. In part, this is due to the fact that a wide variety of manufacturing facilities and operational practices exist, including single-product and multiproduct manufacturing, using traditional segregation strategies with separate rooms for specific operations that may use stainless steel or disposable equipment to more modern ballroom-style operations that use mostly disposable equipment (i.e., pre- and postviral clearance manufacturing operations are not physically segregated by walls). Further, consensus is lacking around basic definitions and approaches related to facility segregation. For example, given that several unit operations provide assurance of virus clearance during downstream processing, how does one define pre- and postviral clearance and at which point(s) should a viral segregation barrier be introduced? What is a "functionally closed" system? How can interventions be conducted so that the system remains functionally closed? How can functionally closed systems be used to adequately isolate a product stream and ensure its safety? To address these issues, the member companies of the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) have conducted a facility segregation project with the following goals: define "pre- and postviral clearance zones" and "pre- and postviral clearance materials"; define "functionally closed" manufacturing systems; and identify an array of facility segregation approaches that are used for the safe and effective production of recombinant biologics as well as plasma products. This article reflects the current thinking from this collaborative endeavor.LAY ABSTRACT: Operations in biopharmaceutical manufacturing are segregated to ensure that the final product has not been contaminated with adventitious viruses, another fraction of the same product, or with another product from within the same facility. Yet there is no consensus understanding of what appropriate facility segregation looks like. There are a wide variety of manufacturing facilities and operational practices. There are existing facilities with separate rooms and more modern approaches that use disposable equipment in an open ballroom without walls. There is also no agreement on basic definitions and approaches related to facility segregation approaches. For example, many would like to claim that their manufacturing process is functionally closed, yet exactly how a functionally closed system may be defined is not clear. To address this, the member companies of the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) have conducted a project with the goal of defining important manufacturing terms relevant to designing an appropriately segregated facility and identifying different facility segregation approaches that are used for the safe and effective production of recombinant biologics as well as plasma products.

Optical Detection of Degraded Therapeutic Proteins.

The quality of therapeutic proteins such as hormones, subunit and conjugate vaccines, and antibodies is critical to the safety and efficacy of modern medicine. Identifying malformed proteins at the point-of-care can prevent adverse immune reactions in patients; this is of special concern when there is an insecure supply chain resulting in the delivery of degraded, or even counterfeit, drug product. Identification of degraded protein, for example human growth hormone, is demonstrated by applying automated anomaly detection algorithms. Detection of the degraded protein differs from previous applications of machine-learning and classification to spectral analysis: only example spectra of genuine, high-quality drug products are used to construct the classifier. The algorithm is tested on Raman spectra acquired on protein dilutions typical of formulated drug product and at sample volumes of 25 µL, below the typical overfill (waste) volumes present in vials of injectable drug product. The algorithm is demonstrated to correctly classify anomalous recombinant human growth hormone (rhGH) with 92% sensitivity and 98% specificity even when the algorithm has only previously encountered high-quality drug product.

Unexplained gastrointestinal bleed due to arteriobiliary fistula after percutaneous liver biopsy.

We represent a case of a 54-year-old male who presented to the emergency department with right upper quadrant abdominal pain and melena three weeks after percutaneous liver biopsy. He was found to have anemia secondary to an upper gastrointestinal hemorrhage, unresponsive to multiple blood transfusions. Angiography later revealed an arteriobiliary fistula with contrast extravasation entering the duodenum. The fistula was successfully embolized and the patient was discharged without complication. This report demonstrates the importance in considering a vascular intrahepatic fistula in patients with right upper quadrant abdominal pain after remote liver biopsy.