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Demonstrating “Abdala” Subunit Vaccine Thermostability Study

By Mabel Izquierdo, Yassel Ramos, Lourdes Costa, Rodolfo Valdés, Yamila Martínez, Mónica Bequet-Romero, Vladimir Besada, Gerardo García, Galina Moya, Glay Chinea, Jennifer Rojas, José Marcelo, Ivan Andujar, Joaquín González, Mareysis Ruiz, Yurisleydis Aldama, Marta Ayala, Jorge Valdés, and Miladys Limonta
Volume 21, Open Access (September 2022)

From a regulatory standpoint, vaccine stability must be demonstrated, along with the prediction of stability during temperature excursions, before a vaccine can be approved for use in humans.

In this work, Abdala subunit vaccine thermostability was studied under thermal stress conditions (2–8°C [control], 25°C, 37°C, 45°C, and 60°C) for 15 days. Molecular integrity of the vaccine active pharmaceutical ingredient was monitored by SDS-PAGE, immunoblotting, RP-HPLC, mass spectrometry, and circular dichroism spectroscopy analysis. While functionality was monitored by immunogenicity assay, inhibition of binding between receptor-binding domain (RBD) and receptor, angiotensin converting enzyme 2 (ACE2), and RBD/ACE2 binding assay.

Results showed that no degradation, loss of disulfide bridges, nor modifications of secondary structure of the RBD molecule were detected at 25°C and 37°C. Moreover, high titers (1:48,853-1:427,849) of anti-RBD-specific mouse antibodies were detected with the ability to inhibit, to different degrees, the binding between RBD/ACE2.

In conclusion, the Abdala subunit vaccine is stable under thermal stress and storage conditions, which has an advantage over non-subunit vaccines previously approved or currently in development against COVID-19. The demonstrated high stability of this vaccine is a key factor in ensuring vaccine effectiveness, extending immunization coverage with fewer production runs, simplifying immunization logistics, and reducing cold chain-associated costs.

Izquierdo M, Ramos Y, Costa L, Valdés R, Martínez Y, Bequet-Romero M, Besada V, García G, Moya G, Chinea G, Rojas J, Marcelo J, Andujar I, González J, Ruiz M, Aldama Y, Ayala M, Valdés J, Limonta M. BioProcess J, 2022; 21.

Posted online September 15, 2022.




Study of a Single-Use Stirred Tank Bioreactor for Manufacturing a Therapeutic Protein in a High Cell Density Microbial Fermentation

By Angus Thompson and Shaunah Rutter
Volume 20, Open Access (Nov 2021)

Stirred tank single-use bioreactors (SUBs) have been widely adopted for production of biopharmaceuticals such as monoclonal antibodies in mammalian cell culture. However, they are seldom used for commercial production of biologics with microbial fermentation. SUBs offer time-saving advantages because they do not require significant downtime for cleaning and sterilization, so finding a SUB that can perform well with high cell density microbial fermentation processes has the potential to increase the number of production runs. Therefore, for this study, a His-tagged protease inhibitor was chosen as a model protein to demonstrate that the Sartorius Biostat STR® MO, a SUB recently developed for microbial fermentation, is suited for recombinant protein production by high cell density Escherichia coli fermentation processes.

At 50 L scale, the SUB achieved good process control and allowed an oxygen uptake rate (OUR) of up to 240 mmoles/L/h. The fermentation runs produced up to 5.8 g/L of the soluble recombinant protein and a dry cell weight of >60 g/L at the end of fermentation. Additionally, the SUB showed a similar fermentation profile when compared with data from parallel runs in 15 L sterilise-in-place (SIP) vessels using identical media and process parameters. This study indicates that with a minimum investment of capital resources, stirred tank SUBs could be used in pilot-scale manufacturing with high cell density microbial fermentations to potentially shorten the timelines and costs of advancing therapeutic proteins to clinic.

Thompson A, Rutter S. Study of a single-use stirred tank bioreactor for manufacturing a therapeutic protein in a high cell density microbial fermentation. BioProcess J, 2021; 20.

Posted online November 5, 2021.


Robust and Scalable GMP Manufacturing of Chondrocyte Cell Therapy for Cartilage Regeneration

By Vinayak Virupaksh Kedage, Rajesh Pratap Singh, Parvathi Chandran, Vidya Jadhav, Sujata Shinde, and Satyen Sanghavi
Volume 20, Open Access (Nov 2021)

Cell therapy has emerged as a promising technology that involves implanting live cells to replace/repair and restore normal function of damaged tissue. Autologous chondrocyte implantation (ACI) has been proven effective for the regeneration of articular cartilage in defective cartilage tissue. The process starts with the collection of healthy tissue from an eligible patient, then isolation and expansion of desired cells in vitro under good manufacturing practice (GMP) conditions, qualification before release of the final cell product, and finally, implantation into the patient. The promise to deliver autologous cell therapies has its own challenges in robust and reproducible manufacturing. To commercialize a cell therapy, it is imperative that a robust and scalable manufacturing process is set up that is consistent, in terms of quality and quantity, in order to deliver the intended therapeutic effect.

We analysed the manufacturing parameters of over 100 cartilage samples that were used to deliver our proprietary, commercialized autologous cell therapy. The paper addresses the most cited challenges in the manufacturing of autologous cell therapies and describes a robust process of in vitro human chondrocyte cell culture. Also included are key factors in manufacturing for attaining a high-quantity and quality product for articular cartilage regeneration.

Kedage VV, Singh RP, Chandran P, Jadhav V, Shinde S, Sanghavi S. Robust and scalable GMP manufacturing of chondrocyte cell therapy for cartilage regeneration. BioProcess J, 2021; 20.

Posted online November 1, 2021.


Optimization of a Conventional Glycosylation Analytical Method Using Machine Learning and Experimental Design

By Eliza Yeung and Philip Ramsey
Volume 20, Open Access (Oct 2021)

Glycosylation is one of the most common post-translational modifications in mammalian-expressed biologics, and is considered to be a critical quality attribute of therapeutic glycoproteins. Due to its biological relevance, physiochemical assessment on the glycosylation profile is always important to the success of a drug development initiative. This article describes the combination of experimental design and machine learning techniques applied to characterize and optimize a conventional, non-derivatized glycoprofiling method on glycans derived from a human immunoglobulin using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Two independent experimental designs, a 16-run definitive screening design (DSD) and a 28-run central composite design (CCD), were incorporated with a machine learning technique known as “self-validating ensemble modeling (SVEM)” and used to build predictive models for four chromatographic responses. We show that the predictive models created using SVEM on the DSD data reliably predicted the behavior of the chosen responses when applied to CCD validation data. This demonstrates that the DSD is an efficient alternative to the larger, traditional CCD in which the combination of experimental design and machine learning can effectively characterize and optimize analytical methods.

Yeung E, Ramsey P. Optimization of a conventional glycosylation analytical method using machine learning and experimental design. BioProcess J, 2021; 20.

Posted online October 15, 2021.


Separation of Empty and Full Adeno-Associated Virus Capsids from a Weak Anion Exchanger by Elution with an Ascending pH Gradient at Low Ionic Strength

By Pete Gagnon, Blaž Goričar, Sara Drmota Prebil, Hana Jug, Maja Leskovec, and Aleš Štrancar
Volume 20, Open Access (Oct 2021)

Separation of empty and full AAV8 capsids was achieved during their elution from a weak anion exchanger with an ascending pH gradient at low conductivity. Experimental data suggest elution was mediated by loss of positive charge from the exchanger. The method produced a full capsid peak with fewer empty capsids than elution of a strong anion exchanger with a salt gradient. Elution of the weak exchanger by sodium chloride gradients or by pH gradients in the presence of sodium chloride gave inferior separation performance. Pre-elution of empty capsids with a pH step allowed full capsids to be eluted by salt without compromising separation. Loading at intermediate pH prevented empty capsid binding and enabled step elution of full capsids in a physiological buffer environment.

Gagnon P, Goričar B, Drmota Prebil S, Jug H, Leskovec M, Štrancar A. Separation of empty and full adeno-associated virus capsids from a weak anion exchanger by elution with an ascending pH gradient at low ionic strength. BioProcess J, 2021; 20.

Posted online October 11, 2021.


A Novel, Risk-based Approach for Predicting the Optimum Set of Process and Cell Culture Parameters for Scaling Upstream Bioprocessing

By Adrian Stacey, Jochen Scholz, and Sinyee Yau-Rose
Volume 20, Open Access (September 2021)

The ability to scale a cell culture effectively and efficiently, from lab to manufacturing, is critical to maximizing productivity whilst minimizing the risk of run failures and delays that can cost millions of dollars per month. The task of scaling well, however, is still considered to be a challenge by many upstream scientists, and this can be an exercise in trial and error. Traditionally, scaling has most often been performed using arithmetic in a spreadsheet and/or simple “back of an envelope” calculations. For some, it may even come in the form of support from a team of data scientists using advanced analytical software. This dependency on what some consider to be complex mathematics or statistics has resulted in the common consideration of using just one scaling parameter at a time, one scale at a time.

However, it is difficult to determine easily or optimally, from the start, whether a process successfully transfers across scales based on only one process parameter, at one scale. In this article, we describe the benefits of using a risk-based approach to scaling, and the development of a software scaling tool known as BioPAT® Process Insights for predictive scale conversion across different bioreactor scales. BioPAT Process Insights can be used to consider multiple parameters and across multiple scales simultaneously, from the start of a scaling workflow. We briefly describe how it was used in a proof-of-concept scale-up study to allow a faster, more cost-effective process transfer from 250 mL to 2000 L. In summary, using BioPAT Process Insights, in conjunction with a bioreactor range that has comparable geometry and physical similarities across scales, has the potential to help biopharma manufacturing facilities reach 2000 L production-scale volumes with fewer process transfer steps, saving both time and money during scale-up of biologics and vaccines.

Stacey A, Scholz J, Yau-Rose S. A novel, risk-based approach for predicting the optimum set of process and cell culture parameters for scaling upstream bioprocessing. BioProcess J, 2021; 20.

Posted online September 28, 2021.


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