Integration of high-throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi-subunit vaccine antigen.

Mammalian cell line generation typically includes stable pool generation, single cell cloning and several rounds of clone selection based on cell growth, productivity and product quality criteria. Individual clone expansion and phenotype-based ranking is performed initially for hundreds or thousands of mini-scale cultures, representing the major operational challenge during cell line development. Automated cell culture and analytics systems have been developed to enable high complexity clone selection workflows; while ensuring traceability, safety, and quality of cell lines intended for biopharmaceutical applications. Here we show that comprehensive and quantitative assessment of cell growth, productivity, and product quality attributes are feasible at the 200-1,200 cell colony stage, within 14 days of the single cell cloning in static 96-well plate culture. The early cell line characterization performed prior to the clone expansion in suspension culture can be used for a single-step, direct selection of high quality clones. Such clones were comparable, both in terms of productivity and critical quality attributes (CQAs), to the top-ranked clones identified using an established iterative clone screening approach. Using a complex, multi-subunit antigen as a model protein, we observed stable CQA profiles independently of the cell culture format during the clonal expansion as well as in the batch and fed-batch processes. In conclusion, we propose an accelerated clone selection approach that can be readily incorporated into various cell line development workstreams, leading to significant reduction of the project timelines and resource requirements.

Formulation Design, Optimization and In Vivo Evaluations of an α-Tocopherol-Containing Self-Emulsified Adjuvant System using Inactivated Influenza Vaccine.

α-Tocopherol has been used as an immune supplement in humans, as an emulsion adjuvant component in several veterinary vaccines as well as an immunomodulatory component of AS03, an emulsion adjuvant that was used in an H1N1 pandemic vaccine (Pandemrix). AS03 is manufactured using microfluidization and high-pressure homogenization. Such high energy and complex manufacturing processes make it difficult and expensive to produce emulsion adjuvants on a large scale, especially in developing countries. In this study we have explored simpler, comparatively inexpensive methods, to formulate emulsion adjuvants containing α-tocopherol, that have the potential to be made in any well-established scale-up facility. This might facilitate producing and stock-piling adjuvant doses and therefore aide in pandemic preparedness. We used design of experiment as a tool to explore incorporating α-tocopherol into self-emulsified systems containing squalene oil and polysorbate 80. We created novel self-emulsified adjuvant systems (SE-AS) and evaluated their potency in vivo in BALB/c mice with inactivated quadrivalent influenza vaccine (QIV) and tested the cellular and humoral immune responses against the four vaccine strains.

Development of a high-throughput Alamar blue assay for the determination of influenza virus infectious dose, serum antivirus neutralization titer and virus ca/ts phenotype.

FluMist is an intranasal influenza live vaccine containing two Influenza A strains (currently H1N1 and H3N2) and one B strain (Yamagata or Victoria lineage). Characterization of the vaccine requires determination of the median tissue culture infectious dose (TCID(50)) titer, serum antivirus neutralization titer and vaccine cold adapted/temperature sensitive (ca/ts) phenotype. Visual cytopathic effect (CPE) readings are used widely in viral assays, but these are subjective and labor intensive. In response to the need for an efficient, inexpensive and high-throughput assay, a 96-well microplate assay was developed that uses Alamar blue dye staining as a replacement for CPE observation in the determination of influenza virus infectious dose, serum antivirus neutralization titer and virus ca/ts phenotype. Relative operating characteristic curves verified that there was a clear distinction between the fluorescence readings of the Alamar blue stained CPE positive and CPE negative wells. Virus titer was determined by use of both Alamar blue staining and CPE-based TCID(50) assays for wild-type and FluMist influenza vaccine strains as well as a plasmid-rescued influenza FluMist A strain containing a H5N1 derived hemmaglutinin and neuramidinase. Correlation of the two assays was measured by regression analysis and resulted in R(2) values of 0.814 (Influenza A), 0.983 (Influenza B) and 1.000 (H5N1), respectively. Serum microneutralization as well as virus ca/ts phenotype assays also showed a high concordance between readings based on CPE observation and Alamar blue staining. The Alamar blue dye assay is user friendly, environmentally safe and sensitive. Also, it is adaptable to automation, which could provide a high-throughput platform for analysis of pre-clinical and clinical samples.

Development of Electrochemiluminescent Serology Assays to Measure the Humoral Response to Antigens of Respiratory Syncytial Virus.

Sensitive and precise serology assays are needed to measure the humoral response to antigens of respiratory syncytial virus (RSV) following natural infection or vaccination. We developed and evaluated a collection of electrochemiluminescent (ECL) serology assays using four RSV antigens (F, N, Ga and Gb). To assess the merits of ECL technology, the four ECL serology assays were evaluated using a well-characterized "gold standard" panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (≥65 years old). The combined results from the four ECL assays demonstrated good concordance to the "gold standard" diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population.

Application of flow cytometry for rapid bioburden screening in vaccine virus production.

Sensitive and timely detection of bioburden in presterile filtration product in aseptic processing of biologics is a critical parameter for microbial control and assurance of final product sterility. An application of automated flow cytometry system was developed for rapid microbial assessment and in-process control in vaccine virus production. In order to minimize the background signal caused by the components of the chicken egg substrate sample matrix, a sample processing method to clear somatic cell debris was included. The sample processing and the automated analysis take approximately 5 to 7 min per test sample and the method provides objective results in real time, enabling uninterrupted processing. The flow cytometry method was compared with the standard aerobic plate count method using tryptic soy agar in a parallel study of 1566 independent production-scale samples. The method was further characterized by spike recovery of five model bacterial organisms in representative sample matrix. In comparison to the culture method, the flow cytometry method was shown to be 96.2% sensitive and 98.2% specific for the detection of bioburden at a level of sensitivity suitable for the process stage requirement with the advantage of a nearly instantaneous time to result. LAY ABSTRACT: In-process bioburden control in the manufacturing of biopharmaceuticals is essential for final product sterility and integrity. In manufacturing contexts where an in-process hold time is infeasible or in cases where uninterrupted processing is desired, conventional culture-based bioburden detection methods cannot be used, as they require significant time to results that may not fit within the time constraints. In this case study we demonstrate the use of flow cytometry as an alternative rapid method that provides real-time results to enable uninterrupted processing.