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A quality- by- design approach for the implementation of a manufacturing license change using a qualified scale- down process model
Get PDFInfluenza vaccines are required to be re formulated every year to account for antigenic drift with recommendations coordinated by the World Health Organisation. This results in a short development cycle of only six months to be able to choose and characterise a suitable reassortant of a recommended strain and proceed to commercial manufacturing of the influenza antigen and vaccine product. Due to the ongoing healthcare crisis brought about the Covid-19 pandemic, the demand for influenza vaccines has increased rapidly requiring vaccine manufacturers to be able to meet this demand and be the first to market in the season. This has necessitated the implementation of novel approaches to increase antigen and vaccine product yield in a rapid and yet robust process development.
One of the potential yield improvements was the introduction of an optimised quantity of Hydrocortisone solution in the egg-based platform process at the inoculum stage for the Influenza A strains, which was already introduced and in use for Influenza B strains. In this presentation, we demonstrate the implementation of a scale-down modelling to support process changes and their subsequent regulatory approval.
To facilitate the implementation of this change to the manufacturing license with various regulatory bodies, we devised a protocol to produce representative antigen batches at scale-down (i.e., 1% scale from a qualified area to the commercial batches) at higher and lower Hydrocortisone input on a variety of Influenza A strains. A total of 11 batches executed with various hydrocortisone inputs and Influenza A seasonal strains showed that the resulting antigen met internal drug substance batch release specifications and showed yield increase of 9-24% across various seasonal Influenza A strains, which could potentially be higher at the commercial scale.
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Novel purification strategies for Influenza neuraminidase-VLPs
Get PDFConventional Influenza virus vaccines reduce the risk of flu illness by between 40% and 60% among the overall population. They are all standardized based on the influenza hemagglutinin (HA) content and focus on the induction of HA-specific neutralizing antibodies. However, in contrast to natural infection, seasonal vaccination fails to induce an efficient immune response to the Influenza virus neuraminidase (NA), the second most abundant viral glycoprotein. A potential solution to this problem would be to supplement current vaccines with a correctly folded, tetrameric recombinant NA protein, or alternatively, with bio-nanoparticles that display the tetrameric NA on an outer membrane. Vaccines that incorporate standardized amounts of NA might lead to broader and longer-lasting protection against influenza infection.
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Maximizing viral titer yield at harvest through metabolic process analytical technology (PAT)
Get PDFThis work pertains to the optimization of enterovirus production using MRC5 cultured on microcarriers within a bioreactor. This enterovirus, like other lytic viruses, has a rapid decay rate within a production batch, such that a 30% loss of potency is observed per day. Therefore, to maximize the yield of infectious product from the bioreactor, harvest needs to be timed to maximize the amount of viral production while minimizing the decay. Viral potency assays have slow turnaround times relative to a production batch, making an online process analytical technology (PAT) critical to maximize titers. In pursuit of an online method for tracking viral titer, three different PAT-enabled streams were investigated: dissolved oxygen (DO), viable cell volume (VCV), and oxygen uptake rate (OUR). DO monitoring was the simplest and leverages the ubiquitous DO trends of production, however it remains scale and gassing strategy dependent. Dual-frequency capacitance measurements were utilized to calculate VCV and thereby quantify the magnitude and timing of massive cell lysis that was correlated in time with peak viral potency. OUR, which quantifies the amount of oxygen being consumed per cellular volume, leverages both capacitance and DO measurements (in addition to oxygen mass balances pertaining to the gassing strategy) to provide a holistic scale-independent metabolic PAT readout. The sharp increase we observe in OUR prior to its decline due to cell lysis appears to be related to increased oxygen demand during viral production—this sharp increase precedes peak viral potency and peak specific productivity in our process. Data generated by our PAT tools—DO, VCV, OUR— were compared to potency and specific productivity trends across 22 batches. In this talk, we will discuss the utility and application of the tools, repeatability of our models across datasets, and the strengths and weaknesses of each model.
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Analytical development to support manufacturing of a sustainable vaccine against Invasive Nontyphoidal Salmonellosis
Get PDFGVGH is developing a candidate trivalent Salmonella vaccine to fight invasive nontyphoidal Salmonellosis (iNTS) and typhoid fever, especially aimed for sub-Saharan Africa to impact disease burden and to reduce anti-microbial resistance spread. This trivalent vaccine may be the only viable option for a sustainable iNTS vaccine in sub-Saharan Africa over the separate administration of Typhoid Conjugate Vaccines (TCV) and a vaccine against iNTS.
GVGH generated the iNTS-TCV formulation by combining the GMMA technology for the iNTS components, S. Typhimurium (STm) and S. Enteritidis (SEn) GMMA adsorbed on Alhydrogel, and the Vi-CRM197 glycoconjugate, originally developed by GVGH and recently WHO prequalified as TCV TYPHIBEV by Biological E Ltd (Hyderabad, India).
A set of analytical methods to support the vaccine lot release and characterization have been developed by GVGH. In particular, to quantify the key active ingredients of iNTS components a competitive ELISA-based method (FAcE, Formulated Alhydrogel competitive ELISA assay) has been setup and characterized in terms of specificity, accuracy and precision. Vi component is instead characterized by means of HPAEC-PAD method, able to specifically identify and quantify the total polysaccharide in the final drug product. With regard to safety assessment, a Monocyte Activation Test (MAT) has been developed as to monitor the intrinsic pyrogenicity of GMMA-based vaccines and applied as surveillance test for the Phase 1 clinical lot, with the plan to set release criteria based on clinical experience.
In vivo potency assay has been set to characterize the immunogenicity of vaccine lots in comparison to freshly formulated material at the time of release and during real-time stability. A significant antibody response to each of the active ingredients of the trivalent vaccine is raised in mice and assessed by Parallel Line Assay.
Overall, the applied analytical panel and the results support the development of an iNTS-TCV vaccine as a viable option for a sustainable iNTS vaccine in sub-Saharan Africa
Development of a universal group 2 influenza virus vaccine using chimeric hemagglutinin constructs
Get PDFThe stalk domain of the hemagglutinin (HA) has become the prime target for universal influenza virus vaccine development in the last few years. Unlike the HA head domain, the immunosubdominant stalk domain is conserved to a higher level within each influenza virus HA group. Sequential vaccination with chimeric HA (cHA) vaccine constructs consisting of the same HA stalk and exotic head domains has proven to re-direct the immune response towards the stalk domain. This vaccination concept provides the basis for the development of more broadly cross-protective vaccines that are less affected by antigenic drift and shift, one of the main drawbacks of currently marketed influenza vaccines.
Most influenza virus vaccines are licensed as inactivated split vaccines. They are manufactured based on HA content with little to no information and standardization of neuraminidase (NA) content. Virus inactivation is generally performed with alkylating agents such as formalin (FA) or β-propiolactone (βPL), rendering the virus unable to infect or replicate. Though safe, whole inactivated virus vaccines can be highly reactogenic. Virus splitting with detergents like sodium deoxycholate (SDCO) and Triton X-100 (TX-100), which dissociate the virus into smaller parts while maintaining a good immunogenicity profile, are typically employed. To date, there are several studies assessing the effect of a variety of inactivating and splitting agents on influenza viruses, but little is known about the impact of combining these agents on HA stalk conformation and NA activity.
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Process development for a flexible vaccine vector platform based on recombinant life virus
Get PDFVaccines are one of the most important, safe and efficient interventions to protect people from illness, disability and death. In recent years several new viral outbreaks where no vaccines are currently available were reported worldwide. Therefore, the development of flexible processes for the production of vaccines is urgently needed. This project aims at developing a platform process for the production of different viral vaccines. The core technology is based on the fact that large recombinant genes coding for selected, foreign antigens can be inserted into the genome of a well-established virus vaccination vector. The vaccine delivers the selected antigens directly to macrophages and dendritic cells, the most potent and effective antigen-presenting cells, thereby triggering a specific immune response to the selected antigens. As a replicating vector, the vaccine continuously expresses antigens even after immunization. This setup results in a powerful, antigen-focused immune response, which is expected to confer long-term immunity.
The challenges in production process design for such a vaccine are the establishment of a robust cell expansion and infection strategy as well the development of efficient downstream processing methods including several chromatography principals, ultra-diafiltration and employment of bio recognition principles. The implementation of a meaningful monitoring/characterization concept furthermore serves as a basis for reliable in-process control strategies
Accurate and precise viral quantification for rapid vaccine development in- process production monitoring using Radiance® Laser Force Cytology\u3csup\u3eTM
Get PDFThe biopharmaceutical world is evolving rapidly, bringing with it the need for technologies to support this fast-paced and changing environment. Trends in biomanufacturing are moving towards shortened development cycles as companies balance increased productivity requirements with the goal of reducing costs while at the same time ensuring production consistencies are met and batch out of specification (OOS) and failure events are minimized. LumaCyte’s Radiance® instrument using Laser Force Cytology™ (LFC), a combination of advanced optics and microfluidics to rapidly analyze single cells based upon their intrinsic biochemical and biophysical cellular properties and without the need for antibodies or labels. Subtle cellular changes can be precisely captured with Radiance’s automated workflow enabling new capabilities for measuring real-time product quality attributes to support R&D, process development and manufacturing needs across the biopharmaceutical industry. In this poster, LumaCyte demonstrates how tedious infectivity assays such as plaque and TCID50 can be replaced by Radiance’s rapid viral infectivity quantification assay to provide significant shorter time to result (TTR), reduced labor, and improved data quality and consistency. In addition, the bioproduction of vaccines, viral vectors or VLPs can be monitored in real-time, enabling rapid optimization of key processes and increasing process knowledge. As a result, product yield can be increased using the same inputs and the likelihood of OOS events can be reduced. Radiance applications in oncolytic virus research and neutralization assays are presented as well. Overall, LFC delivers faster TTR and improved data quality for vaccine analytics from R&D to manufacturing
The next generation of fibroblast-based vaccine development
Get PDFChicken embryo fibroblasts (CEF) and diploid cells have a long history in vaccine production since their isolation in the 1960s at the Wistar Institute (WI-38 cells) as well as the Medical Research Council (MRC-5 cells). The cells quickly became adopted for a number of vaccines: varicella zoster (VZV), MMR, yellow fever, polio, hepatitis A, rotavirus, rabies, Marek\u27s disease, and dengue virus. Most of these vaccine processes were developed with classical media supplemented with Fetal Bovine Serum (FBS). The Hayflick limit of diploid cells restricted their adaptation to a serum-free process. While some of the vaccines such as polio and rabies have been transitioned to Vero cells, several vaccines continue to be manufactured with CEF and human diploid cells. Currently, FBS from Australia and New Zealand are utilized for the highest level of patient safety for human vaccines. However, this supply of serum is challenged by two factors: growth of existing vaccines to improve global access and the development of new gene therapies that require FBS. In order to reduce dependency on serum, we initiated a medium development program. Using metabolite analysis and DOE, we have developed a serum-reduced growth medium and a serum-free virus production medium for MRC-5 and other fibroblast cells. With a serum reduction of 90-100%, the growth medium can support direct recovery from thaw and adaptation-free expansion, resulting in performance that is comparable to classical medium with 10% serum. We confirmed virus production with VZV and vesicular stomatitis virus in MRC-5 cells as well as Marek’s disease virus in CEFs and demonstrate a higher specific productivity. By switching to a low serum process, vaccine manufacturers can reduce production and purification costs, and increase product consistency and safety
Protective complex oxide film formation in multi-component ultra-high temperature carbides during plasma jet exposure
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Processing of high entropy carbide based ceramics
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