Alice Bernier

Improved high-performance liquid chromatographic method in the analysis of adenovirus particles

We developed a HPLC method on a novel continuous bed matrix (UNO Q, Bio-Rad) for the direct quantification of adenoviral type 5 (Ad5) particles produced in 293S Human Embryonic Kidney cells and compared this with an existing HPLC method on a conventional ion-exchange resin (Resource Q, Pharmacia). The 293S cell extract contained large amounts of DNA. This contaminated the viral peak on the Resource Q column and only after Benzonase treatment was it possible to quantify the viral particles in the cell extract. In contrast, the virus peak on the UNO Q column was resolved from the DNA which eliminates the need for pretreatment of the sample with Benzonase. Cross-analysis of the Ad5 fraction from the UNO Q column using a size-exclusion HPLC column revealed no additional contaminating peaks. We conclude that the purity of the Ad5 virus peak on the continuous bed matrix UNO Q column was superior to the purity of the virus on the conventional Resource Q column, which is essential for reliable quantification.

Validation of a high-performance liquid chromatographic assay for the quantification of adenovirus type 5 particles.

An anion-exchange-high-performance liquid chromatography (AE-HPLC) method for the quantification of adenovirus type 5 (Ad5) total particles was validated according to performance criteria of precision, specificity, linearity of calibration and range, limit of detection, limit of quantification, accuracy and recovery. The viral particles were detected by absorbance at 260 nm using photodiode array detector (PDA). Cesium chloride (CsCl) purified Ad5 and lysate samples were used for the validation of the method. Relative standard deviations (RSDs) for the inter-day, intra-day precision and reproducibility for both the lysate and the Ad5 standard were less than 10 and 2% for the peak area and retention time, respectively. The method was specific for Ad5 which was eluted at 8.0 min. The presence of DNA does not affect the recovery of Ad5 particles for accurate quantification. Based on the error in prediction to be less than 10%, the working range was established between 2 x 10(10) and 7 x 10(10) VP/ml with correlation coefficient of 0.99975, standard deviation of 6.14 x 10(9) VP/ml and a slope of 3.04 x 10(5) VP/ml. The recovery of the method varied between 88 and 106% in all of the lysate samples investigated which is statistically similar to 100% recovery at 95% confidence interval.

Virus-like Particle and Viral Vector Production Using the Baculovirus Expression Vector System/Insect Cell System

The ability to make a large variety of virus-like particles (VLPs) has been successfully achieved in the baculovirus expression vector system (BEVS)/insect cell system. The production and scale-up of these particles, which are mostly sought as candidate vaccines, are currently being addressed. Furthermore, these VLPs are being investigated as delivery agents for use as therapeutics. Recently, adeno-associated viral (AAV) vectors, which can be potentially used for human gene therapy, have been produced in insect cells using three baculovirus vectors to supply the required genes. The use of host insect cells allows mass production of VLPs in a proven scaleable system. This chapter focuses on the methodology, based on the work done in our lab, for the production of AAV-like particles and vectors in a BEVS/insect cell system.

An efficient process for the purification of helper-dependent adenoviral vector and removal of helper virus by iodixanol ultracentrifugation.

The preparation of large amount of purified helper-dependent adenoviral vector material is hampered by the lack of development of downstream processes with proven records on separation and recovery efficiencies. In order to facilitate the use of clinical-grade helper-dependent virus material for large-scale in vivo studies, a three-step purification scheme consisting of (1) an anion-exchange chromatography for initial capturing of virus, (2) a shallow iodixanol density gradient ultracentrifugation for the removal of helper virus from helper-dependent virus, and (3) a size-exclusion chromatography for the removal of iodixanol and residual protein contaminants as a polishing step was developed. The use of a fast iodixanol density ultracentrifugation step was highly effective in separating infectious helper-dependent virus from contaminating helper virus. The overall downstream processing scheme gave 80% infectious particle yield. The contamination ratio of helper virus in the helper-dependent virus preparation are reduced from 2.57 to 0.03% corresponding to a reduction of helper virus by factors of 85 by two iodixanol purification steps. It was also demonstrated that size-exclusion chromatography is an excellent step for the removal of iodixanol and polishing of the final helper-dependent virus preparation.

Establishment and validation of new complementing cells for production of E1-deleted adenovirus vectors in serum-free suspension culture.

E1-deleted adenovirus vectors (AdV) are important gene transfer vehicles for gene therapy and vaccination. Amplification of AdV must take place in cells that express the adenovirus E1A and E1B genes. Sequence homology between AdV and the E1 genes integrated within the complementing cells should be minimal to reduce the odds of generating replication-competent adenovirus (RCA). The present study describes the establishment of AdV complementing cells constructed by stable transfection of the minimal E1A and E1B genes into human lung carcinoma (A549). Because some transgene products can be cytotoxic, the cells were engineered to stably express the repressor of the cumate-switch (CymR) to silence transgene transcription during vector growth. For regulatory compliance and to facilitate the scale-up, the resulting complementing cells (SF-BMAdR) were adapted to serum-free suspension culture. The best clone of SF-BMAdR produced AdV carrying an innocuous transgene to the same level as 293 cells, but titers were better for AdV carrying transgene for a cytotoxic product. Elevated titers were maintained for at least two months in suspension culture in the absence of selective agent and the cells did not produce RCA. Because of their advantageous properties, SF-BMAdR cells should become an important tool for developing large-scale production processes of AdV for research and clinical applications.

Rapid and reliable quantification of reovirus type 3 by high performance liquid chromatography during manufacturing of Reolysin

Reolysin, a human reovirus type 3, is being evaluated in the clinic as an oncolytic therapy for various types of cancer. To facilitate the optimization and scale-up of the current process, a high performance liquid chromatography (HPLC) method has been developed that is rapid, specific and reliable for the quantification of reovirus type 3 particles. Using an anion-exchange column, the intact virus eluted from the contaminants in 9.78 min at 350 mM NaCl in 50mM HEPES, pH 7.10 in a total analysis time of 25 min. The virus demonstrated a homogenous peak with no co-elution of other compounds as analyzed by photodiode array analysis. The HPLC method facilitated the optimization of the purification process which resulted in the improvement of both total and infectious particle recovery and contributed to the successful scale-up of the process at the 20 L, 40 L and 100 L production scale. The method is suitable for the analysis of crude virus supernatants, crude lysates, semi-purified and purified preparations and therefore is an ideal monitoring tool during process development and scale-up.

Optimization and scale-up of cell culture and purification processes for production of an adenovirus-vectored tuberculosis vaccine candidate.

Tuberculosis (TB) is the second leading cause of death by infectious disease worldwide. The only available TB vaccine is the Bacille Calmette-Guerin (BCG). However, parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. AdAg85A, an adenoviral vector expressing the mycobacterial protein Ag85A, is a new tuberculosis vaccine candidate, and has shown promising results in pre-clinical studies and phase I trial. This adenovirus vectored vaccine is produced using HEK 293 cell culture. Here we report on the optimization of cell culture conditions, scale-up of production and purification of the AdAg85A at different scales. Four commercial serum-free media were evaluated under various conditions for supporting the growth of HEK293 cell and production of AdAg85A. A culturing strategy was employed to take advantages of two culture media with respective strengths in supporting the cell growth and virus production, which enabled to maintain virus productivity at higher cell densities and resulted in more than two folds of increases in culture titer. The production of AdAg85A was successfully scaled up and validated at 60L bioreactor under the optimal conditions. The AdAg85A generated from the 3L and 60L bioreactor runs was purified through several purification steps. More than 98% of total cellular proteins was removed, over 60% of viral particles was recovered after the purification process, and purity of AdAg85A was similar to that of the ATCC VR-1516 Ad5 standard. Vaccination of mice with the purified AdAg85A demonstrated a very good level of Ag85A-specific antibody responses. The optimized production and purification conditions were transferred to a GMP facility for manufacturing of AdAg85A for generation of clinical grade material to support clinical trials.

Cell Culture Tracking by Multivariate Analysis of Raw LCMS Data

Liquid chromatography mass spectrometry (LCMS) is a powerful technique that could serve to rapidly characterize cell culture protein expression profile and be used as a process monitoring and control tool. However, this application is often hampered by both the sample proteome and the LCMS signal complexities as well as the variability of this signal. To alleviate this problem, culture samples are usually extensively fractionated and pretreated before being analyzed by top-end instruments. Such an approach precludes LCMS usage for routine on-line or at-line application. In this work, by applying multivariate analysis (MA) directly on raw LCMS signals, we were able to extract relevant information from cell culture samples that were simply lyzed. By using the recombinant adenovirus production process as a model, we were able to follow the accumulation of the three major proteins produced, identified their accumulation dynamics, and draw useful conclusions from these results. The combination of LCMS and MA provides a simple, rapid, and precise means to monitor cell culture.

292. Towards Large-Scale Manufacturing of Adeno-Associated Virus by Transient Transfection of HEK293 Suspension Cells in a Stirred Tank Bioreactor Using Serum-Free Medium

Adeno-Associated Virus (AAV) vectors showing safety profile in phase I clinical trials and its ability to transduce gene expression in various tissues have made it a vector of choice for gene delivery. There are different modes of AAV vector production and each has advantages and disadvantages. Here we demonstrated that the production of AAV by transient transfection in a serum-free medium using NRCs patented cGMP compliant human embryonic kidney HEK293 cell line (clone HEK293SF-3F6) adapted for growth in suspension can be readily scaled-up in stirred tank bioreactors. We employed triple-plasmid / polyethylenimine (PEI) based transient transfection technique. As a proof of concept, we demonstrated that nine serotypes of AAV (AAV-1 to AAV-9) encoding GFP can be produced by our cell line HEK293SF with yields of about 1E+13 genome-containing particles per liter (Vg/L). Depending on the serotypes 4-30% of AAV is present in the supernatant of the cell culture at 48hpt. The presence of plasmids and plasmid polyplexes that were not taken up by the cells or were not brought into the cell nucleus were removed by Iodixanol-ultracentrifugation method and Benzonase treatment before analyzing by real-time PCR. About 25% loss in genome containing viral particle counts were observed by Iodixanol purification method based on infectivity assay. Productions of AAV2 and AAV6 encoding GFP were demonstrated in 3L stirred tank bioreactors. Purification scheme was based on column chromatography - a scalable process. Different chromatography media, such as cation exchanger, anion exchanger and hydrophobic interaction chromatography, were tested with each AAV serotypes for their ability to adsorb and elute efficiently. The purification scheme was then adopted by integrating best chromatography medium and sequence dependent upon the AAV serotype in use. We demonstrated the purification scheme for AAV2 based on ion-exchange and hydrophobic interaction chromatography steps. The SDS-PAGE showed the purity of the final product and the presence of three capsid proteins VP1, VP2 and VP3 on Western blot corresponding to the only three bands present in the final product on SDS-PAGE. To extend the storage life of AAV we explored lyophilization technique to study the stability of AAV2 and AAV6 under lyophilized conditions. The AAV2 and AAV6 were stable for over 40 weeks based on infectivity assay. We demonstrated the scalability of the process up to 45L. Productions tested in 20 and 500 mL cultures in shake flasks were scaled up in 2 and 45L cultures (in 3- and 60-L stirred tank bioreactors, respectively). The volumetric yields and purification recoveries were comparable at all of these production scale levels demonstrating scalability of transient transfection at even larger scale is possible to generate material necessary for dosages required for gene therapy application.

Optimization and scale-up of cell culture and purification processes for production of an adenovirus-based tuberculosis vaccine

Background Tuberculosis (TB) is the second leading cause of death by infectious disease worldwide. About 1.4 million people die of TB each year. Parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. AdAg85A adenovirus, a new promising tuberculosis vaccine candidate, has been studied with mouse, guinea pig, goat and cow animal models, and was shown to be effective against Mycobacterium tuberculosis (Mtb) infection [1]. A phase I trial on Ad5Ag85A adenovirus was also conducted, which demonstrated that AdAg85A adenovirus was safe and highly immunogenic [2]. To further evaluate the efficacy of this vaccine and reduce the cost of this promising vaccine candidate, a feasible and cost-effective large-scale cell culture production process had to be developed for manufacturing large quantities of AdAg85A adenovirus required for further clinical trials. Furthermore, the process had to be designed to meet all requirements for industrialization and commercialization of this vaccine candidate. Here we report our study on optimization of cell culture conditions, scale up of AdAg85A adenovirus production in 60L bioreactor and purification of the AdAg85A adenovirus at different scales. The optimized conditions for AdAg85A adenovirus production and purification were transferred to a GMP facility for manufacturing of AdAg85A adenovirus for further clinical trials. Materials and methods Four commercial serum-free cell culture media (SFM4HEK-293 and SFM4Transfx-293 from HyClone; Adenovirus Expression Medium (AEM) and CD 293 from Life Technologies), were evaluated for supporting the growth of HEK293SF-3F6 cell in suspension and also for the production of AdAg85a adenovirus in 125 mL shake flask cultures under various experimental conditions. The production of the AdAg85A adenovirus was then scaled up to 3L controlled bioreactor under the optimized conditions obtained from the shake flask experiment, further validated in a 60L bioreactor. Purification of the AdAg85A adenovirus was accomplished through many different steps. Some of the critical steps include cell lysis, benzonase treatment, Q-Sepharose HP anion exchange chromatography for capture of adenovirus/purification, Capto Core 700 multimodal chromatography for polishing, concentration and diafiltration into formulation buffer. The purification processes were also scaled up from 3L to 60 L production scale.