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Practical Considerations in Using an Equivalence Approach to Establish Lot Release Limits for Vector Dose

By Nancy Sajjadi and Janice Callahan
Volume 22, Open Access (November 2023)

To demonstrate that a dose-determining assay is fit for purpose, the measurement uncertainty associated with a reported release test result must be suitably small. The establishment of a corresponding product specification is inextricably linked to the tolerance for error in assigning a dose value for a vector lot. By adopting an equivalence-based lot release model which includes a total error approach to assay qualification, specific testing strategies can be evaluated quantitatively for dose error and lot release decision risks throughout the drug development process. This article aims to reinforce how the concepts tied to an equivalence-based lot release model are interrelated and applied in practice. It provides in-depth explanations of fundamental concepts and clarifies common misunderstandings for quality control, quality assurance, and regulatory affairs personnel held accountable for decisions made in vector dose assignment and product lot release.

Sajjadi N, Callahan JD. Practical considerations in using an equivalence approach to establish lot release limits for vector dose. BioProcess J, 2023; 22.

Posted online November 17, 2023.


The Impact of Process Closure on Biomanufacturing Risk

By Jeffery N. Odum, CPIP
Volume 22, Open Access (September 2023)

“Closed system.” The term itself appears deceptively simple. However, the definition of a closed system, its implementation, and its impact on biomanufacturing has been anything but straightforward.

The journey toward implementing closed systems spans over 20 years. The concept of closed systems was introduced in January 2000 with the draft issue of ICH Q7. Since then, other industry guidance documents emerged, defining and supporting process/system closure as a primary means of risk mitigation to meet the baseline requirement of protecting the product, as defined in cGMP.

Presently, global regulatory agencies recognize three distinct definitions of a closed system. These definitions, found in EU Annex 1, EU Annex 2, and the PIC Annex 2A, all focus on product protection where the product is not exposed to the immediate room environment during manufacturing. This is where the journey begins.

Odum JN. The impact of process closure on biomanufacturing risk. BioProcess J, 2023; 22.

Posted online September 12, 2023.


Article Preview: Practical Considerations in Using an Equivalence Approach to Establish Lot Release Limits for Vector Dose

By Nancy Sajjadi and Janice Callahan
Volume 22, Open Access (June 2023)

The approval of several gene therapy products and gene-modified cell therapies over the last five years has led to increasing numbers of investigational new drug applications (INDs) using adeno-associated and lentiviral vectors. However, these successes have been tempered by the risks of dose-related toxicities. The therapeutic window for a product is derived from pre-clinical and clinical dose response models, which assume statistically that measurements of dose are exact. Whether vector is administered directly or used as a critical reagent to prepare a gene-modified cellular product, the assignment of a label concentration to a vector batch is critical for establishing consistency of product used in preclinical and clinical development.

Sajjadi N, Callahan J. Article preview: practical considerations in using an equivalence approach to establish lot release limits for vector dose. BioProcess J, 2023; 22.

Posted online June 19, 2023.


Gamma-Immunoglobulin Response Characterization, in COVID-19 Convalescent Patients, Against the Spike Protein S2 Subunit with Eight Linear Peptides for Monoclonal Antibody Generation

By Airela Llamo, Daily Hernández, Cristina García, Marcos González, Williams Ferro, Hilda Garay, David Diago, Abel Fajardo, Luis Espinosa, Sigifredo Padilla, Leonardo Gómez, Glay Chinea, and Rodolfo Valdés
Volume 22, Open Access (March 2023)

The SARS-CoV-2 spike protein S2 subunit plays an essential role in the virus-host cell membrane fusion process. Therefore, the subject of this study was to characterize the gamma-immunoglobulin (IgG) response, in a group of COVID-19 convalescent patients, against the S2 subunit with eight linear peptides to generate a monoclonal antibody (mAb) against the immunodominant linear peptide to be used for therapeutic and diagnostic purposes. Results of antibody percentages against assessed linear peptides were 100% for A21P73, A21P74, A21P75, A21P76, M20P51, M20P65, M20P83, and 66.7% for M20P85. Plasma samples were also used for purifying IgG to corroborate specificity against the same linear peptides, where results reproduced those applying plasmas directly to ELISA-plates. Within these peptides, A21P75 was chosen as immunodominant (100% of recognition with higher absorbance). The A21P75 linear peptide showed poor immunogenicity in mice (1:4000–8000 after four doses), allowing the generation of a CB.HS2A21P75 hybridoma for mAb production that recognized the A21P75 linear peptide with middle-to-high affinity constant (Kaff) (0.8×108 M-1).

This study concludes that the A21P75 linear peptide is the assessed immunodominant linear peptide for this COVID-19 convalescent patient group. This peptide is located in the HR1 region that plays an important role in SARS-CoV-2 host cell membrane fusion process and is highly conserved between SARS-CoV-2 and SARS-CoV. Thus, due to CB.S2A21P75 mAb specificity and Kaff, it might be the proper reagent to study inhibition of virus-host cell membrane fusion, and as a diagnostic reagent for coronavirus. Finally, the combination of A21P75 linear peptide with other peptides (e.g., receptor binding domain [RBD]) could be suitable reagents for the development of vaccines and therapeutic antibodies with virus infection-blocking capacity.

Llamo A, Hernández D, García C, González M, Ferro W, Garay H, Diago D, Fajardo A, Espinosa L, Padilla S, Gómez L, Chinea G, Valdés R. Gamma-immunoglobulin response characterization, in COVID-19 convalescent patients, against the spike protein S2 subunit with eight linear peptides for monoclonal antibody generation. BioProcess J, 2023; 22.

Posted online March 6, 2023.


Human IgG Fc Production Through Methanol-Free Pichia pastoris Fermentation

By Ying Yang, Knut Madden, and Ma Sha
Volume 21, Open Access (November 2022)

Nowadays, therapeutic monoclonal antibodies (mAbs) are predominantly produced with mammalian cell culture systems such as those using Chinese hamster ovary (CHO) cells. Efforts are underway to reduce the costs of this process to meet the increasing global demand in biopharmaceuticals; meanwhile, cheaper and faster expression systems are being investigated as alternatives. The yeast, Pichia pastoris, has become a substantial workhorse for recombinant protein production. However, the N-linked glycosylation in P. pastoris, namely high mannose glycosylation, is significantly different from that in CHO or other mammalian cells, including human cells. In this study, a SuperMan5 strain of P. pastoris was constructed using Pichia GlycoSwitch® technology to successfully produce a more mammalian-like immunoglobulin G (IgG) fragment crystallizable (Fc), which showcases the potential of P. pastoris as a next-generation mAb production platform. Importantly, in this study, a strong methanol-independent promoter, PUPP, was applied, which only requires glycerol feeding for protein production. Most P. pastoris promoters used for protein expression are derived from genes in the methanol metabolism pathway, creating safety concerns due to the flammable nature of methanol, especially at large scale. Here, a fed-batch SuperMan5 P. pastoris fermentation was carried out in which methanol induction, as well as its affiliated safety risks, were eliminated. Overall, this study provides insights into the development of safe and cost-effective industrial mAb production approaches independent of mammalian cell culture.

Yang Y, Madden K, Sha M. Human IgG Fc production through methanol-free Pichia pastoris fermentation. BioProcess J, 2022; 21.

Posted online November 21, 2022.


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.


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