Rapid In-Process Monitoring of Lentiviral Vector Particles by High-Performance Liquid Chromatography.

Lentiviral vectors (LVs) are a popular gene delivery tool in cell and gene therapy and they are a primary tool for ex vivo transduction of T cells for expression of chimeric antigen receptor (CAR) in CAR-T cell therapies. Extensive process and product characterization are required in manufacturing virus-based gene vectors to better control batch-to-batch variability. However, it has been an ongoing challenge to make quantitative assessments of LV product because current analytical tools often are low throughput and lack robustness and standardization is still required. This paper presents a high-throughput and robust physico-chemical characterization method that directly assesses total LV particles. With simple sample preparation and fast elution time (6.24 min) of the LV peak in 440 mM NaCl (in 20 mM Tris-HCl [pH 7.5]), this ion exchange high-performance liquid chromatography (IEX-HPLC) method is ideal for routine in-process monitoring to facilitate the development of scalable and robust LV manufacturing processes. Furthermore, this HPLC method is suitable for the analysis of all in-process samples, from crude samples such as LV supernatants to final purified products. The linearity range of the standard curve is 3.13 × 108 to 1.0 × 1010 total particles/mL, and both the intra- and inter-assay variabilities are less than 5%.

Particle quantification of influenza viruses by high performance liquid chromatography.

The influenza virus continuously undergoes antigenic evolution requiring manufacturing, validation and release of new seasonal vaccine lots to match new circulating strains. Although current production processes are well established for manufacturing seasonal inactivated influenza vaccines, significant limitations have been underlined in the case of pandemic outbreaks. The World Health Organization called for a global pandemic influenza vaccine action plan including the development of new technologies. A rapid and reliable method for the quantification of influenza total particles is crucially needed to support the development, improvement and validation of novel influenza vaccine manufacturing platforms. This work presents the development of an ion exchange-high performance liquid chromatography method for the quantification of influenza virus particles. The method was developed using sucrose cushion purified influenza viruses A and B produced in HEK 293 suspension cell cultures. The virus was eluted in 1.5 M NaCl salt with 20 mM Tris-HCl and 0.01% Zwittergent at pH 8.0. It was detected by native fluorescence and the total analysis time was 13.5 min. A linear response range was established between 1 × 10(9) and 1 × 10(11) virus particle per ml (VP/ml) with a correlation coefficient greater than 0.99. The limit of detection was between 2.07 × 10(8) and 4.35 × 10(9) whereas the limit of quantification was between 6.90 × 10(8) and 1.45 × 10(10)VP/ml, respectively. The coefficient of variation of the intra- and inter-day precision of the method was less than 5% and 10%. HPLC data compared well with results obtained by electron microscopy, HA assay and with a virus counter, and was used to monitor virus concentrations in the supernatant obtained directly from the cell culture production vessels. The HPLC influenza virus analytical method can potentially be suitable as an in-process monitoring tool to accelerate the development of processes for the manufacturing of influenza vaccines.

Transient gene expression in serum-free suspension-growing mammalian cells for the production of foot-and-mouth disease virus empty capsids.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.

Development and validation of a HPLC method for the quantification of baculovirus particles.

A HPLC method using an anion exchange column was developed for the quantification of baculovirus particles. To properly detect the virus eluting from the column, a nucleic acid dye was used to amplify the signal projected by the virus. The viral genome was labeled by incubating the virus with SYBR Green I at 37°C for a minimum of 1h. The virus was specifically eluted from the contaminants in 8.9 min at a NaCl concentration of 480 mM NaCl (in 20 mM Tris-HCl, pH 7.5). The total run time of the method was 25 min. The method resulted in a linear response from 1×10(8) to 5.0×10(10)viral particles (VP/ml). The detection limit was 3.0×10(7) and the quantification limit was 1×10(8)VP/ml. The intra-assay precision was <10% for both purified and crude virus preparations whereas the inter-assay precisions were <5% and <10% for purified and crude virus preparations, respectively. The recovery/accuracy of the method ranged from 78 to 101%. This method is a robust monitoring tool to facilitate research activities with baculovirus vector and accelerate development of baculovirus-based processes for manufacturing of biologics.

Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing.

Cell culture processes offer an attractive alternative to conventional chicken egg-based influenza vaccine production methods. However, most protocols still rely on the use of adherent cells, which makes process scale-up a challenging issue. In this study, it is demonstrated that the HEK-293 human cell line is able to efficiently replicate influenza virus. Production in serum-free suspension of HEK-293 cultures resulted in high titers of infectious influenza viruses for different subtypes and variants including A/H1, A/H3 and B strains. After virus adaptation and optimization of infection conditions, production in 3-L bioreactor resulted in titers of up to 10(9)IVP/mL demonstrating the scale-up potential of the process.

Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures.

BACKGROUND: Lentiviral vectors (LV) offer several advantages over other gene delivery vectors. Their potential for the integration and long-term expression of therapeutic genes renders them an interesting tool for gene and cell therapy interventions. However, large-scale LV production remains an important challenge for the translation of LV-based therapeutic strategies to the clinic. The development of robust processes for mass production of LV is needed. METHODS: A suspension-grown HEK293 cell line was exploited for the production of green fluorescent protein-expressing LV by transient polyethylenimine (PEI)-based transfection with LV-encoding plasmid constructs. Using third-generation packaging plasmids (Gag/Pol, Rev), a vesicular stomatitis virus G envelope and a self-inactivating transfer vector, we employed strategies to increase volumetric and specific productivity. Functional LV titers were determined using a flow cytometry-based gene transfer assay. RESULTS: A combination of the most promising conditions (increase in cell density, medium selection, reduction of PEI-DNA complexes per cell, addition of sodium butyrate) resulted in significantly increased LV titers of more than 150-fold compared to non-optimized small-scale conditions, reaching infectious titers of approximately 10(8) transducing units/ml. These conditions are readily scalable and were validated in 3-liter scale perfusion cultures. CONCLUSIONS: Our process produces LV in suspension cultures and is consequently easily scalable, industrially viable and generated more than 10(11) total functional LV particles in a single bioreactor run. This process will allow the production of LV by transient transfection in sufficiently large quantities for phase I clinical trials at the 10-20-liter bioreactor scale.

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.

Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.

We have developed new packaging cell lines (293SF-PacLV) that can produce lentiviral vectors (LVs) in serum-free suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2(S)-M2) of the tetracycline (Tet) switch, was established first. We next generated clones stably expressing the Gag/Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G). Expression of Rev and VSV-G was tightly regulated by the cumate and Tet switches. Our best packaging cells produced up to 2.6 x 10(7) transducing units (TU)/ml after transfection with the transfer vector. Up to 3.4 x 10(7) TU/ml were obtained using stable producers generated by transducing the packaging cells with conditional-SIN-LV. The 293SF-PacLV was stable, as shown by the fact that some producers maintained high-level LV production for 18 weeks without selective pressure. The utility of the 293SF-PacLV for scaling up production in serum-free medium was demonstrated in suspension cultures and in a 3.5-L bioreactor. In shake flasks, the best packaging cells produced between 3.0 and 8.0 x 10(6) TU/ml/day for 3 days, and the best producer cells, between 1.0 and 3.4 x 10(7) TU/ml/day for 5 days. In the bioreactor, 2.8 liters containing 2.0 x 10(6) TU/ml was obtained after 3 days of batch culture following the transfection of packaging cells. In summary, the 293SF-PacLV possesses all the attributes necessary to become a valuable tool for scaling up LV production for preclinical and clinical applications.

Validation of a high-performance liquid chromatographic assay for the quantification of Reovirus particles type 3.

An anion exchange high-performance liquid chromatography (HPLC) method for the quantification of human Reovirus type 3 particles was validated according to the performance criteria of precision, specificity, linearity of calibration and working range, limits of detection and quantification, accuracy and recovery. Samples taken at various stages of Reovirus purification were used for the validation of the method. The method was specific for Reovirus which eluted around 9.8min without interference from any other component in the sample. Reovirus can be detected between 0.32E+12 and 2.10E12VP/mL by the proposed method that has the correlation coefficient of linearity equal to 0.9974 and the slope of linearity equal to 5.74E-07 area units/(VPmL).

High yield purification of functional baculovirus vectors by size exclusion chromatography.

Recombinant baculoviruses carrying mammalian expression cassettes or "BacMam" are promising gene delivery vehicles shown to transduce mammalian cells efficiently both in vitro and in vivo. These viruses are vectors of choice because they are non-pathogenic; able to accommodate large foreign DNA inserts and can be produced at high titers. Hence, the demand for pure and functional baculovirus vectors for gene delivery experiments is anticipated in the future. The main goal of this work is to develop a simple and efficient process to purify recombinant baculovirus derived from Autographa californica multiple nucleopolyhedrovirus from a culture supernatant by size exclusion chromatography. The final yields obtained for total and infectious particles were 1.39 x 10(11) and 1.02 x 10(10) and recoveries of 25% and 24%, respectively. The virus was purified from the majority of the protein contaminants as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Negative stain electron microscopy demonstrated that >95% of the purified virus was intact particles with shape like rod and average diameter and length of 60 and 266 nm, respectively. Transduction of 293 human embryonic kidney cells by a purified GFP-expressing BacMam at a multiplicity of transduction of 200 resulted in 36% positive cell population.

Retroviral vector production using suspension-adapted 293GPG cells in a 3L acoustic filter-based perfusion bioreactor.

Recombinant retroviruses are now an established tool for gene delivery. Presently they are mainly produced using adherent cells. However, due to the restrictive nature of adherent cell culture, this mode of production is hampered by low cell-specific productivity and small production units. The large-scale production of retroviral vectors could benefit from the adaptation of retrovirus packaging cell lines to suspension culture. Here, we describe the ability of a 293 packaging cell line to produce retroviral vectors in suspension culture at high titer. Adherent 293GPG cells, producing a Moloney Murine Leukemia Virus (MoMLV) retrovirus vector pseudotyped with the vesicular stomatitis virus G (VSVG) envelope protein and expressing a TK-GFP fusion protein, were adapted to suspension culture in calcium-free DMEM. At a cell density similar to adherent cell culture, the suspension culture produced retroviral vector consistently in the range of 1 x 10(7) infectious viral particles/mL (IVP/mL), with a specific productivity threefold higher than adherent culture. Furthermore, at the same medium replacement frequency, the suspension producer cells could be cultured at higher density than their adherent counterparts, which resulted in virus titer of 3-4 x 10(7) IVP/mL at 11.0 x 10(6) cells/mL. This corresponds to a 10-fold increase in viral concentration compared to adherent cells. The capacity to up scale the retroviral vector production was also demonstrated by performing a 2 VVD perfusion culture for 9 days in a 3L Chemap bioreactor. The combination of suspension and perfusion led to a 20-fold increase in maximum virus productivity compared to the adherent culture.

High-performance liquid chromatographic total particles quantification of retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein.

A novel and rapid method for the total particles quantification of murine leukemia virus derived retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein was developed using high performance liquid chromatography. Virus particles were detected by absorbance at 260 nm and quantified using a calibration curve generated from highly purified and concentrated viral stock characterized by negative stain electron microscopy. The method requires Benzonase digestion and concentration of the supernatant prior to analysis. The virus eluted in 12.55 min at a flow rate of 1 mL/min in 20 mM Tris-Cl, pH 7.4 + 1.1 M NaCl. The limits of detection and quantification of this assay were 4.71 x 10(8) and 1.57 x 10(9) viral particles/mL, respectively. Linearity was between 3.0 x 10(9) and 1.0 x 10(11) viral particles/mL with a correlation coefficient of 0.9923 and a slope of 6 x 10(-6). The assay precision was <5% and <10% for intra- and inter-day analysis, respectively. This assay was used for the total particles quantification of a 7-day, large-scale perfusion culture production of a retroviral vector grown in 293 cells expressing the beta-galactosidase gene.

Size-exclusion chromatography purification of high-titer vesicular stomatitis virus G glycoprotein-pseudotyped retrovectors for cell and gene therapy applications.

Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped replication-defective retroviral particles are pantropic and amenable to concentration to high titer by ultracentrifugation. These features have allowed development of effective retroviral transduction protocols for stem cells in vitro as well as for tissue engineering in vivo. However, retroparticle ultracentrifugation protocols will also copellet cellular and subcellular debris released from retroviral producer cell lines during vector manufacture. We have analyzed concentrated vector preparations by chromatography and have found that a significant amount of genomic DNA released from producer cells coconcentrates with retroviral particles. In an effort to generate high-purity retroparticle preparations, devoid of subcellular contaminants and contaminating genomic DNA, we have developed a process using size-exclusion chromatography combined with host cell nucleic acid digestion and concentration by ultrafiltration. The procedure allowed for a final recovery of 19 +/- 0.4% infectious viral particles from unfractionated starting material, with an average retroparticle concentration of 7.7 x 10(7) +/- 1.5 x 10(6)/ml. The intact virus is of high purity, >90% as determined by anion-exchange high-performance liquid chromatography. Retroparticle structure appeared intact as determined by negative stain electron microscopy and purified virus was functional and allowed for efficient transduction of primary human bone marrow stromal cells in vitro. In conclusion, we have developed a VSV-G retrovector purification process that can be applied to large-scale retroviral production ideal for cell and gene therapy applications.