Establishing Suspension Cell Cultures for Improved Manufacturing of Oncolytic Adenovirus.

Recent clinical trials have shown the potential of oncolytic adenoviruses as a cancer immunotherapy. A successful transition of oncolytic adenovirus to clinical applications requires efficient and good manufacturing practice compatible production and purification bioprocesses. Suspension cultures are preferable for virus production as they can reduce process costs and increase product quality and consistency. This work describes the adaptation of the A549 cell line to suspension culture in serum-reduced medium validated by oncolytic adenovirus production in stirred tank bioreactor. Cell concentrations up to 3 × 106 cells mL-1 are obtained during the production process. At harvest 1.4 × 1010 infectious particles mL-1 and 6.9 ± 1.1 × 1010 viral genome mL-1 are obtained corresponding to a viral genome: infectious particles ratio of 5.2 (± 1.9): 1 confirming the virus quality. Overall, the suspension characteristics of these A549 cells support an easily scalable, less time-consuming, and more cost-effective process for expanded success in the use of oncolytic viruses for cancer therapy.

Clinical-Grade Oncolytic Adenovirus Purification Using Polysorbate 20 as an Alternative for Cell Lysis.

INTRODUCTION: Oncolytic virus therapy is currently considered as a promising therapeutic approach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach. Triton X-100 is commonly used in cell lysis buffer preparations. The addition of this surfactant in the list of substances with the very high concern of the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation promoted the research for effective alternatives. METHODS: In this work, a purification strategy for oncolytic adenovirus compatible with phase I clinical trials, using an approved surfactant - Polysorbate 20 was developed. The proposed downstream train, composed by clarification, concentration using tangential flow filtration, intermediate purification with anion exchange chromatography, followed by a second concentration and a final polishing step was evaluated for both Triton X-100 and Polysorbate 20 processes. The impact of cell lysis with Polysorbate20 and Triton X-100 for each downstream step was evaluated in terms of product recovery and impurities removal. Overall, 61 ± 4% of infectious viral particles were recovered. Depletion of host cell proteins and ds-DNA was 99.9% and 97.1%, respectively. RESULTS & CONCLUSION: The results indicated that Polysorbate 20 can be used as a replacement for Triton X-100 during cell lysis with no impact on product recovery, potency, and purity. Moreover, the developed process is scalable and able to provide a highly purified product to be used in phase I and II clinical trials.

Efficient chromatographic reduction of ovalbumin for egg-based influenza virus purification.

Vaccination is the most effective prevention strategy to avoid influenza infection and for protection of large populations. The vast majority of influenza vaccines are still produced with allantoic fluid from fertilized chicken eggs. The presence of ovalbumin, which can constitute over 60% of the total protein content in allantoic fluid, can result in severe allergies. Consequently, efficient reduction of ovalbumin is critical during egg based vaccine manufacturing. Here we present Capto Core 700, a novel core bead chromatographic flow through mode resin for removal of ovalbumin and compare it to sucrose zonal gradient ultracentrifugation, which is the industry standard for egg-based vaccine production. The results demonstrate that core bead chromatography is fully comparable to zonal centrifugation in removing ovalbumin to meet regulatory requirements. Furthermore, the scalability and the shorter process times of this method have the potential to significantly improve the productivity and economy for industrial production compared to zonal centrifugation.

Sensitive methods for evaluation of antibodies for host cell protein analysis and screening of impurities in a vaccine process.

BACKGROUND: Host cell proteins (HCP) should be carefully monitored in vaccine production. To achieve a reliable HCP estimation, a mixture of polyclonal antibodies (pAbs) with broad affinity would be of preference. Sensitive evaluations of the pAbs are therefore of value. METHODS: Column purification of specific HCPs with affinity to the anti-HCP pAbs was compared with Western blotting of the anti-HCP pAbs binding to filter bound total lysate. The anti-HCP pAbs were used in an HCP quantification analysis using surface plasmon resonance (SPR). Host cell derived impurities from an influenza vaccine process were analyzed using 2-D DIGE analysis. RESULTS: The Western blotting showed a similar HCP binding pattern of anti-HCP pAbs from immunizations using two adjuvants: CFA/IFA and AbISCO(®). From the column purification of HCPs, total proteins detectable were similar for all anti-HCP pAbs; however the immune response pattern differed significantly for the anti-HCP pAbs from the AbISCO(®) immunization. In the SPR HCP quantification assay the standard curve ranged from 0.3 to 40 µg/ml. The advantage of SPR compared with ELISA was the decreased hands on time and that the sample number was not limiting. The 2-D DIGE showed that most of the HCPs were removed at the clarification and virus capture step. DISCUSSION: Column purification of HCPs with affinity to the anti-HCP pAbs increased the sensitivity of affinity analysis compared with Western blotting and opened the possibility of further analysis. The anti-HCP pAbs did not interact with proteins in the virus; simplifying analysis of process samples using SPR. 2-D DIGE analysis gave a direct study of the impurity profile with the advantage of independence from antibody performance.

Expression, purification and characterization of the recombinant chimeric IgE Fc-fragment opossum-human-opossum (OSO), an active immunotherapeutic vaccine component.

The active vaccine component recombinant chimeric IgE Fc-fragment opossum-human-opossum (OSO) has been expressed in CHO-K1 cells. It contains two identical polypeptide chains with 338 amino acid residues in each chain connected by two disulfide bridges. The cell lines were adapted to suspension culture in a serum-free medium. An expression level of 60 mg/L was obtained after 8 days in a shaking flask at a temperature of 31.5 degrees C. The OSO protein has been purified to homogeneity by a combination of three chromatographic steps. Virus inactivation and reduction by solvent detergent treatment and nano-filtration were included in the process. The residual host cell protein content was less than 50 ng/mg OSO as analyzed by ELISA. Purity was analyzed by SDS-PAGE under reducing and non-reducing conditions and was estimated by densitometry to be above 99.0%. The dimer content was less than 0.1% as estimated by analytical size exclusion chromatography. The molecular mass, as estimated by SDS-PAGE, is 90 kDa. A value of around 74 kDa was calculated from its amino acid composition. This indicates that the protein is heavily glycosylated containing around 18% carbohydrate. Isoelectric focusing in polyacrylamide gel disclosed a ladder type band pattern with pI values in the pH-range 7.0-8.3, indicating a variation in the sialic acid content. The OSO protein is not stable at temperatures above 40 degrees C and at pH values below 4 indicating that virus inactivation by incubating the protein solution at higher temperature or at lower pH is not possible.

Automation of cell line development.

An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used for evaluation of the automation approach. It is shown that the automated procedure is capable of producing cell lines of equal quality to the traditionally generated cell lines in terms of colony detection following transfection and distribution of IgG titer in the screening steps. In a generic fed-batch evaluation in stirred tank bioreactors, IgG titers of 4.7 and 5.0 g/L were obtained for best expressing cell lines. We have estimated that the number of completed cell line development projects can be increased up to three times using the automated process without increasing manual workload, compared to the manual process. Correlation between IgG titers obtained in early screens and titers achieved in fed-batch cultures in shake flasks was found to be poor. This further implies the benefits of utilizing a high throughput system capable of screening and expanding a high number of transfectants. Two concentrations, 56 and 75 muM, of selection agent, methionine sulphoximine (MSX), were applied to evaluate the impact on the number of colonies obtained post transfection. When applying selection medium containing 75 muM MSX, fewer low producing transfectants were obtained, compared to cell lines selected with 56 muM MSX, but an equal number of high producing cell lines were found. By using the higher MSX concentration, the number of cell line development projects run in parallel could be increased and thereby increasing the overall capacity of the automated platform process.

Binding of Ikaros to germline Ig heavy chain gamma1 and epsilon promoters.

Immunoglobulin (Ig) class switching occurs in activated B cells and results in production of antigen-specific IgA, IgE or IgG. It involves a DNA recombination event and is partly regulated by germline (GL) immunoglobulin heavy chain promoters. Ikaros is an abundant nuclear protein expressed in hematopoietic cells. Many different functions have been ascribed to Ikaros, such as transcriptional activation or repression, cell cycle control and tumor suppression. A typical feature of Ikaros is its expression in large clusters in the nucleus of activated lymphocytes. We give evidence that Ikaros can bind to several sites in the germline gamma1 and epsilon immunoglobulin heavy chain promoters, in a cooperative manner. Using a promoter reporter assay, we found evidence that Ikaros can suppress germline gamma1 and epsilon promoter activity in a B cell line. When a mutated non-DNA-binding form of Ikaros was introduced into primary activated B cells by retrovirus transduction, the endogenous Ikaros clusters were disrupted. In spite of this, there was no effect on transcription or Ig class switching. The data are discussed in relation to the different hypotheses for the function of Ikaros.