Adventitious Agents Research Articles

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.

Computational prediction of blend time in a large-scale viral inactivation process for monoclonal antibodies biomanufacturing.

Viral inactivation (VI) is a process widely used across the pharmaceutical industry to eliminate the cytotoxicity resulting from trace levels of viruses introduced by adventitious agents. This process requires adding Triton X-100, a non-ionic detergent solution, to the protein solution and allowing sufficient time for this agent to inactivate the viruses. Differences in process parameters associated with vessel designs, aeration rate, and many other physical attributes can introduce variability in the process, thus making predicting the required blending time to achieve the desired homogeneity of Triton X-100 more critical and complex. In this study we utilized a CFD model based on the lattice Boltzmann method (LBM) to predict the blend time to homogenize a Triton X-100 solution added during a typical full-scale commercial VI process in a vessel equipped with an HE-3-impeller for different modalities of the Triton X-100 addition (batch vs. continuous). Although direct experimental progress of the blending process was not possible because of GMP restrictions, the degree of homogeneity measured at the end of the process confirmed that Triton X-100 was appropriately dispersed, as required, and as computationally predicted here. The results obtained in this study were used to support actual production at the biomanufacturing site.

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.

Rapid In-Process Measurement of Live Virus Vaccine Potency Using Laser Force Cytology: Paving the Way for Rapid Vaccine Development.

Vaccinations to prevent infectious diseases are given to target the body’s innate and adaptive immune systems. In most cases, the potency of a live virus vaccine (LVV) is the most critical measurement of efficacy, though in some cases the quantity of surface antigen on the virus is an equally critical quality attribute. Existing methods to measure the potency of viruses include plaque and TCID50 assays, both of which have very long lead times and cannot provide real time information on the quality of the vaccine during large-scale manufacturing. Here, we report the evaluation of LumaCyte’s Radiance Laser Force Cytology platform as a new way to measure the potency of LVVs in upstream biomanufacturing process in real time and compare this to traditional TCID50 potency. We also assess this new platform as a way to detect adventitious agents, which is a regulatory expectation for the release of commercial vaccines. In both applications, we report the ability to obtain expedited and relevant potency information with strong correlation to release potency methods. Together, our data propose the application of Laser Force Cytology as a valuable process analytical technology (PAT) for the timely measurement of critical quality attributes of LVVs.

Action of Bromelain and Ficin on horse anti Bothrops sp venom Antibodies

The treatment with hyperimmune sera constitute the only specific and effective therapy available against snakebite envenomation, most common in developing countries. Serum quality is an important factor on patient recovery time and in the incidence of death and permanent disability. To date, most sera consist of pepsin digested IgG antibodies harvested from hyperimmune animals. The use of animal derived enzymes, such as pepsin, to digest IgG, constitute a source of adventitious agents and contaminants, such as porcine circovirus. The present study aims to evaluate the use of the plant derived enzymes bromelain and ficin, as an alternative to pepsin. To this purpose, horse serum immunized against Bothrops venoms was purified with caprylic acid and digested with bromelain or ficin. SDS-PAGE results evidence the formation of F(ab)’2 fragments and suggest that a digestion time superior to 8 hours may be required to completely digest the antibodies with bromelain or ficin. F(ab)’2 fragments obtained by digestion with either bromelain or ficin digestion preserved the ability to recognize Bothrops sp. venom in western blotting assays. Therefore, both enzymes are suitable for use in large-scale production, minimizing contamination risks and increasing safety and efficiency of serotherapy treatments.

LABRADOR—A Computational Workflow for Virus Detection in High-Throughput Sequencing Data

High-throughput sequencing (HTS) allows detection of known and unknown viruses in samples of broad origin. This makes HTS a perfect technology to determine whether or not the biological products, such as vaccines are free from the adventitious agents, which could support or replace extensive testing using various in vitro and in vivo assays. Due to bioinformatics complexities, there is a need for standardized and reliable methods to manage HTS generated data in this field. Thus, we developed LABRADOR—an analysis pipeline for adventitious virus detection. The pipeline consists of several third-party programs and is divided into two major parts: (i) direct reads classification based on the comparison of characteristic profiles between reads and sequences deposited in the database supported with alignment of to the best matching reference sequence and (ii) de novo assembly of contigs and their classification on nucleotide and amino acid levels. To meet the requirements published in guidelines for biologicals’ safety we generated a custom nucleotide database with viral sequences. We tested our pipeline on publicly available HTS datasets and showed that LABRADOR can reliably detect viruses in mixtures of model viruses, vaccines and clinical samples.

Proceedings of the 2019 Viral Clearance Symposium, Session 7: Cell Banks and U.S. Pharmacopeia.

Adventitious contamination of a manufacturing process is a major concern for biopharmaceutical manufacturers. This session focused on rapid detection methods for adventitious agents and mitigation methods for upstream media and feeds. In the first session, it was shown that next-generation sequencing (NGS) can be a good rapid alternative to existing methods used to detect adventitious virus contamination. The second session showed that upstream virus barrier filters can robustly remove virus from media and feeds and could be a good alternative to high-temperature short time (HTST) aimed at facility contamination risk mitigation. In the third session, testing for Mycobacterium species was requested by the Chinese health authority for cell banks. In this case, the risk of mycobacterial contamination was minimal for well-characterized cell banks, and robust downstream processes are in place to remove any potential adventitious contaminants such as viruses and bacteria; therefore, it was not needed. In the final session, a review of rapid testing methods for viral detection was discussed as well as the possibility of using these technologies to replace existing methods.

Proceedings of the 2019 Viral Clearance Symposium, Session 8. Conference Summary: Key Discussion and Outcomes, Pending Questions, and Proposed Experiments and Actions.

This report provides a high-level summary of the key outcomes and gaps based on the research presented at the 2019 Viral Clearance Symposium and identifies new areas for future study and improvements. The 2019 conference structure extended the framework from the preceding conferences, focusing on the key gaps and associated developments and including the additional focus areas of facility risk mitigation, modular viral clearance claims, depth filter viral clearance, retrospective analysis of viral clearance and continuous processing, quantitation and analysis of viral clearance, viral inactivation by detergents and low pH, viral filtration mechanisms, viral filtration of media, viral detection by next-generation sequencing, and ways to improve the efficiency of the overall adventitious agent strategy.

Expression systems for viral vector production: advantages of the Sf9 baculovirus system and simple solutions to address its specific analytical challenges

<p class="focus-visible" data-focus-visible-added="">The gene therapy landscape has exploded in the last few years, bringing a multitude of viral vectors to the clinic. At the forefront of this evolution is the application of lentivirus, adeno and adeno-associated virus (AAV), and plasmid-based therapies to genetic diseases of all types. With each program that progresses through the clinic, the body of manufacturing and clinical knowledge grows – and so does the availability of regulatory guidance. Adeno-associated virus (AAV) is increasingly popular, and the baculovirus-Sf9 platform has been established as a promising alternative to mammalian cell-based methods. However, the Sf9 baculovirus production system poses some unique analytical challenges. Here, we will discuss two of these issues – quantifying residual DNA and detecting the adventitious agent Sf-rhabdovirus – and solutions developed by Thermo Fisher Scientific designed to meet regulatory guidelines and ensure product quality and safety when utilizing the baculovirus-Sf9 platform for AAV production.

Viral adventitious agent detection using laser force cytology: Intrinsic cell property changes with infection and comparison to in vitro testing.

Adventitious agent testing in biomanufacturing requires assays of broad detection capability to screen for as many infectious agents as possible. The current gold standard for general infectious adventitious virus screening is the in vitro assay in which test articles are cultured onto a panel of different cell lines and observed for cytopathic effect (CPE). However, this assay is inherently subjective due to the nature of visual observation of cell morphology and labor and time intensive, requiring highly trained personnel to identify CPE. Laser force cytology (LFC) is an alternative, automated analytical method that uses a combination of optical and fluidic forces along with imaging to objectively and quantitatively assess CPE in cell culture. Importantly, because LFC uses no labels or antibodies, the assay is appropriate for general adventitious agent testing. Using LFC, changes in cellular features associated with virally infected cells were identified using principal component analysis. Using these features of infected cells, the sensitivity and earliness of detection with LFC was directly compared with the in vitro assay for a diverse panel of viruses incubated with chinese hamster ovary (CHO), Vero, and Medical Research Council cell strain 5 (MRC-5) cells. LFC detected viral infection with a sensitivity equal to the in vitro assay on average, but in certain virus and cell combinations including mouse minute virus (MMV) and reovirus 3 in CHO cells, detection was 4 days earlier and for MMV, the limit of detection was 10-fold lower. Overall, these results demonstrate the ability of LFC to serve as a biopharmaceutical adventitious agent testing methodology with sensitivity equivalent to the in vitro assay, but in an objective and automated manner.

Efficacy of minute virus of mice (MVM) inactivation utilizing high temperature short time (HTST) pasteurization and suitability assessment of pasteurized, concentrated glucose feeds in Chinese hamster ovary (CHO) cell expression systems.

There is a growing need to provide effective adventitious agent mitigation for high risk upstream cell culture raw materials used for the production of biologics. It is also highly important in the growing fields of cell and gene therapies. Glucose is a critical raw material necessary for effective cell growth and productivity; however, glucose is the highest risk animal‐origin‐free raw material for viral contamination, and often the highest risk raw material in the upstream process as more companies move to chemically defined media. This study examines the efficacy of utilizing High Temperature Short Time (HTST) pasteurization for inactivation of physiochemically resistant, worst‐case parvovirus using a bench‐scale HTST system. We demonstrated approximately six log inactivation of Minute Virus of Mice (MVM) in concentrated glucose feeds without impacting the subsequent performance of the glucose in a Chinese Hamster Ovary (CHO) expression system.

Revisiting mouse minute virusinactivation 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.

An adventitious agent-free clonal cell line that is highly susceptible to foot -and-mouth disease virus

Porcine LFBKαVβ6 cells have been successfully used for diagnostics and propagation of all FMDV serotypes/subtypes. Unfortunately, after initial characterization, these cells showed contamination with bovine viral diarrhea virus (BVDV), a non-cytopathic adventitious agent. Persistent infection with BVDV could interfere with diagnostic tests and, also prevent consideration for other uses, i.e., vaccine production. In this study, we developed a three-prong methodology to completely remove BVDV from LFBKαVβ6 cells. Combined treatment with siRNA against BVDV NS5A, porcine interferon alpha and ribavirin resulted in the elimination of BVDV, as determined by immunohistochemistry analysis, quantitative RT-PCR and RNA sequencing. Importantly, elimination of BVDV from LFBKαVβ6 did not affect FMDV growth and plaque phenotype from different serotypes isolated and propagated in the clean cell line, newly named MGPK αVβ6-C5. Additionally, isolation of FMDV from field oro-pharyngeal samples, was successful at the same sensitivity as in BVDV-contaminated LFBKαVβ6 cells. Our results identified a direct method to efficiently eliminate BVDV from porcine cells without altering FMDV permissiveness, diagnostic value, or potential for use in vaccine production. Furthermore, these cells may provide an improved platform for diagnostics and propagation of other viruses of interest in the veterinary field and the virology community at large.

Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids

Rapid diagnostics of adventitious agents in biopharmaceutical/cell manufacturing release testing and the fight against viral infection have become critical. Quantitative real-time PCR and CRISPR-based methods rapidly detect DNA/RNA in 1 h but suffer from inter-site variability. Absolute quantification of DNA/RNA by methods such as digital PCR reduce this variability but are currently too slow for wider application. Here, we report a RApid DIgital Crispr Approach (RADICA) for absolute quantification of nucleic acids in 40-60 min. Using SARS-CoV-2 as a proof-of-concept target, RADICA allows for absolute quantification with a linear dynamic range of 0.6–2027 copies/μL (R2 value > 0.99), high accuracy and low variability, no cross-reactivity to similar targets, and high tolerance to human background DNA. RADICA's versatility is validated against other targets such as Epstein-Barr virus (EBV) from human B cells and patients' serum. RADICA can accurately detect and absolutely quantify EBV DNA with similar dynamic range of 0.5–2100 copies/μL (R2 value > 0.98) in 1 h without thermal cycling, providing a 4-fold faster alternative to digital PCR-based detection. RADICA therefore enables rapid and sensitive absolute quantification of nucleic acids which can be widely applied across clinical, research, and biomanufacturing areas.

PCR Testing of Media Placed in Soiled Bedding as a Method for Mouse Colony Health Surveillance.

Rodent colony health surveillance has traditionally been accomplished by testing sentinel animals that have been exposed to soiled bedding from colony animals. Collecting samples from exhaust plenums on ventilated caging systems, followed by PCR analysis, has emerged as another promising method for health surveillance. However, environmental testing at the rack level is not effective for all ventilated rack designs. In this study, we tested whether media placed in soiled bedding is effective in detecting 3 adventitious agents: mouse norovirus (MNV), Helicobacter spp., and fur mites. Soiled bedding was collected from pathogen-positive colony mice and distributed to traditional sentinel mouse cages and mouse-free experimental cages every 1 to 2 wk for static and ventilated cages, respectively. Experimental cages contained 10 flocked swabs ('passive swabs') and 1 piece of filter media. After 90 d, fresh feces, pelage swabs, and blood were collected from the sentinel cages, and the passive swabs and filter media were collected from the experimental cages. Concurrently, 10 additional flocked swabs ('active swabs') were stirred through the cumulated soiled bedding of each experimental cage. Sentinel mice were positive for MNV and Helicobacter spp., but negative for fur mites by pelage swab PCR. All samples from experimental cages were positive for Helicobacter spp. and fur mites in both caging types. For MNV, passive swabs were most effective at detection (100%), followed by active swabs (80% to 100%) and filter media (60% to 80%). These findings suggest that testing media in pooled soiled bedding samples is more effective than traditional sentinel methods for colony health surveillance and is a viable option when sampling at the rack level is ineffective.

Manufacturing freshly cultured umbilical cord-derivedmesenchymal stromal cells (UC-MSCS) for a phase 1, multiple-dose clinical trial for COVID-19-induced acute respiratory distress syndrome (ARDS)

Background & Aim: The Cellular Immuno-Therapy for COVID-19 related ARDS (CIRCA-19) was a phase 1, single site, dose escalation trial using a 3+3+3 design to determine the safety and maximum feasible tolerated dose of intravenously delivered, freshly cultured UC-MSCs. Nine patients, each receiving repeated unit doses of UC-MSCs over 3 consecutive days, were enrolled into 3 dose panels: Panel 1: 25×106 cells/dose (cumulative dose: 75×106 MSCs);Panel 2: 50×106 cells/dose (cumulative dose: 150×106 MSCs);Panel 3: up to 90×106 cells/dose (cumulative dose: 270×106 MSCs). Methods, Results & Conclusion: UC-MSCs were isolated from cords of healthy term pregnancies delivered by C-section. Cords were mechanically and enzymatically digested, and UC-MSCs were propagated in xeno-free conditions for 2 weeks prior to cryopreservation in a cord specific cell bank. One fully validated cell bank was used in CIRCA-19 that was free of adventitious agents (HBV, HCV, HSV1/2, Parvo B19 and Retroviruses), had high viability (>95%) and MSC identity with positive expression (>95%) of CD73, CD90 and CD105 and negative expression ( 45%;DBT = 22h) and enhanced IDO expression (ΔΔCq?>18) when treated with IFN-γ. For the final product, UC-MSCs were thawed, plated and cultured for 24 to 120 h before harvesting to produce a batch of the final drug product formulated as 2.5×106 fresh UC-MSCs/mL suspended in PlasmaLyte A containing 5% Human Albumin, to be infused within 48h. Batches were tested for viability, endotoxin level, ACE-2 expression, tissue factor activity, sterility and mycoplasma. Sixteen batches of UC-MSCs were produced for a total of 41 cell doses (16 doses of 25M;13 doses of 50M;12 doses of 90M cells each). Twenty-seven of the 41 doses (9 doses of 25M, 50M and 90M cells each) were used to treat trial participants. The remaining doses were used for stability studies. All drug products had high viability (> 95%), endotoxin levels of 35;GAPHD Cq: 15±2;no detectable levels by western blotting) and had tissue factor activity levels between 250-310pM. UC-MSC drug product was stable for up to 96h (>80% viability) and had?>90% viability up to 48h in all 3 dose panels. This study demonstrates the feasibility of manufacture and delivery of a multi-dose fresh cell product in an emergent ICU setting.

Sequence-independent single-primer-amplification (SISPA) as a screening technique for detecting unexpected RNA viral adventitious agents in cell cultures

The sequence-independent, single-primer amplification (SISPA) enables the random amplification of nucleic acids, allowing the detection and genome sequencing of different viral agents. This feature of SISPA method provides evidence for application of it in monitoring the presence of adventitious RNA viruses in cell cultures. We evaluated SISPA method for the detection of a challenge RNA virus representing adventitious agent in cell cultures. Besides, by optimizing the SISPA method in our laboratory, we found false-positive results on negative control lanes in electrophoresis gels. To investigate the sources of contamination, false-positive results of SISPA were cloned into Escherichia coli cells, sequenced, and phylogenetically analyzed. This data revealed that the SISPA method can be used as an adjunct method to confirm the absence of unexpected adventitious RNA viruses in cell cultures. The phylogenetic analysis of SISPA contaminant sequences showed that the false-positive results were caused by nucleic acid amplification of commercial cDNA synthesis kit reagents, probably tracing back to expression plasmids and host ribosomal sequences, used for the production of enzymes. Therefore, laboratories using random amplification methods must be constantly aware of the potentials of such contaminations, yielding false-positive results and background noise in the final NGS reads.

Application of machine learning methods to pathogen safety evaluation in biological manufacturing processes.

The production of recombinant therapeutic proteins from animal or human cell lines entails the risk of endogenous viral contamination from cell substrates and adventitious agents from raw materials and environment. One of the approaches to control such potential viral contamination is to ensure the manufacturing process can adequately clear the potential viral contaminants. Viral clearance for production of human monoclonal antibodies is achieved by dedicated unit operations, such as low pH inactivation, viral filtration, and chromatographic separation. The process development of each viral clearance step for a new antibody production requires significant effort and resources invested in wet laboratory experiments for process characterization studies. Machine learning methods have the potential to help streamline the development and optimization of viral clearance unit operations for new therapeutic antibodies. The current work focuses on evaluating the usefulness of machine learning methods for process understanding and predictive modeling for viral clearance via a case study on low pH viral inactivation.

Bioproduction of Pharmaceuticalsbioprocess and the Developing World

Industrial animal cell culture is used to make many life-saving biopharmaceutical proteins, vaccines and cell therapies. Contamination of an industrial animal cell culture with a microorganism, such as a bacteria or virus, may occur through many means, for example, human error, inadequate aseptic protocols within biosafety level 2 (BSL-2) cabinets, failure of a processing step such as steam sterilization, loss of equipment integrity such as a crack in a disposable bioreactor, and/or introduction of a new adventitious agent not susceptible to current removal or inactivation procedures.The term microorganism is synonymous with the common term microbe (adjective microbial). In response to such contamination crises, many firms simultaneously implemented a large number of changes, in emergency mode, without first identifying the source of the problem or thus understanding the likely effectiveness of any given change. Over time, one key change or two typically solved the problem. Sometimes the source of the problem, as well as the key change(s) that actually solved the problem, were identified. Other times, no such clear identifications were made. In nearly all cases, the whole slew of changes were carried forward, even though some were likely ineffective, as well as a waste of time, money and focus. Yet many people in developing countries who could benefit from pharmaceuticals do not receive them. The failure of antiretroviral therapy to reach more than a tiny fraction of people with AIDS in developing countries has attracted widespread publicity, but even medicines that are far cheaper and easier to deliver are not reaching many of the people who need them. More than a quarter of children worldwide and over half of children in some countries do not receive the vaccines that are part of WHO’s Expanded Program on Immunization, although these cost only pennies per dose and require no diagnosis. Three million lives are lost annually as a result (World Bank, 2001a). Only a small fraction of children in poor countries receive the newer hepatitis B and Haemophilus influenzae b (Hib) vaccines, which cost a dollar or two per dose. One in four people worldwide suffer from intestinal worms, although treatments only need to be taken once or twice per year, have virtually no side effects, and cost less than a dollar per year. When asked which viral barriers proved impractical, company representatives had mixed responses. Filtration was characterized by some as expensive, having poor flux properties, or not suitable for use with bulk medium as some important media components were filtered out. Conversely, many interviewees stated that filtration was quite practical for small volumes, including heat-sensitive supplements, as well as hydrophobic additions. While certain respondents described HTST as quite practical and cost effective, others described it as ineffective due to cost, the large space it takes in the plant, and incompatibility with serum and hydrolysed. Since the first public disclosure of a large-scale adventitious agent contamination by Genentech, the topic of viral barriers for upstream processes has become more mainstream. However, while many mid-size or large companies have investigated the implementation of barriers and their associated challenges, some companies remain uncertain about the difficulties they may face if they would like to implement a barrier in upstream cell culture processes.

Recent Advances of Chemical Definition of Cell Culture Media and Excipients for Virus and Viral Vector Manufacturing: A Review

A vital step in the manufacturing of vaccines and viral vector-based cell and gene therapies is virus production. Historically, virus production relies heavily on undefined and highly variable raw material for cell-based platforms to be successful. Yet due to supply chain constraints, poor reproducibility, and safety concerns attributed to potential adventitious agents present in serum and serum-derived proteins, regulatory agencies have encouraged replacement of these components. Recombinant serum proteins, specifically albumin and transferrin, can successfully replace serum-derived proteins in the expansion of VERO cells and subsequent production of multiple virus types. Furthermore, recent successes have demonstrated that recombinant human serum albumin can serve as an effective excipient in final formulations for the stabilization of virus in vaccines. Collectively, chemical definition of both upstream and downstream processes provides numerous advantages and is essential given the importance of virus and viral vector production for clinical applications.