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19 January 2022 by OXGENE

Overcoming the Challenges of Large Scale Plasmid Production

Gene therapy is one of the most exciting fields of biopharma today, but plasmids pose serious challenges for innovation and drug development. WuXi’s acquisition of OXGENE means a fusion of expertise, helping to create an efficient means of developing plasmids on a large scale.

Gene therapy involves inserting a new section of DNA into apatient’s cells. This DNA is delivered by viral vectors, which are usually built from plasmids. Plasmids are small sections ofgenetic material that can be combined to contain all the genes necessary to deliver the gene therapy.

Many challenges associated with manufacturing therapieson a large scale are caused by difficulties when working withplasmids. When manufacturing a plasmid, the final productmust be consistent across batches and must not have altered genetic material or other variations that affect quality. Often, small-scale manufacturing processes using adherent cell require significant expertise and process optimisation to adapt to suspension culture for clinical or commercial use. There are also regulatory and timescale challenges associated with plasmid production which delay drug development.

In 2021, WuXi AppTec acquired OXGENE to become part of WuXi Advanced Therapies. Together, we support innovators all the way from discovery – including optimised vector design and engineering, through large-scale plasmid manufacture and supply– to GMP viral vector and cell therapy manufacture and testing.

Optimising Plasmid Design

To meet the challenge of large-scale production, a plasmid must be able to reliably produce a high yield of viral product. OXGENE have optimised these proprietary plasmids, and the technology used to design them, to meet this need. OXGENE’s adeno-associated virus (AAV) and lentiviral packaging
plasmids are based on proprietary SnapFast™ technology. The system works like ‘molecular building blocks’, using a catalogue of characterised DNA elements that can be inserted into defined locations to allow precise construction of a specific plasmid.

Improved AAV Packaging Plasmids

Most recombinant adeno-associated virus (rAAV) production systems retain the configuration of rep and cap genes found in the viral genome. However, since the cytotoxic and cytostatic effects of rep are well documented, we used SnapFast™ plasmid technology to place these genes under the control of different promoters to express rep at a lower level than cap (2, 3). This resulted in up to an eight-fold increase in viral titre – depending on
rAAV serotype – compared to the native configuration.

AAV production also requires adenoviral help. This can be provided by co-transfection of the core adenoviral helper genes in a third plasmid or by adenoviral infection. The latter generally results in much higher AAV yield, but also significant adenoviral contamination. To improve helper-free AAV production, we’ve simplified the adenoviral helper plasmid. The reduced plasmid size can save costs, as smaller plasmids often grow better than larger ones, giving higher yields and requiring fewer DNA preparations. This minimised helper plasmid also removes additional adenoviral hexon gene sequences, which is regarded favourably by regulators.

Improved Lentiviral Packaging Plasmids

OXGENE’s proprietary third-generation lentiviral packaging system is comprised of four plasmids. The first encodes Gag, a polyprotein that forms the viral core structure, and Pol, the reverse transcriptase. The second encodes the regulatory protein Rev, essential for HIV-1 protein expression. The third, an envelope plasmid, encodes VSV-g which fuses the viral envelope to the host cell membrane. The fourth plasmid is the transfer plasmid, containing the rest of the viral genome and the transgene of interest.

Third-generation plasmids are safer than second-generation systems because gag-pol and rev are kept in different plasmids, so an extra recombination event would be required for a replication competent lentivirus.

However, the enhanced safety features of a third-generation lentiviral packaging plasmid system often come at the cost of viral titre. To combat this, OXGENE have modified the lentiviral packaging plasmids.

We have used a combination of strong viral promoters and hybrid promoters to optimise the expression level of each packaging element and codon optimised vsv-g and rev to increase translation efficiency. We have also enhanced performance of the transfer plasmid by removing non-essential restriction sites, while inserting extra cloning sites. This gives the system more flexibility and improves packaging efficiency and viral titre regardless of the transgene.

Optimising a Custom GOI Plasmid

OXGENE’s expertise in molecular biology, combined with the SnapFast™ plasmid technology, allows optimal collaboration with customers to further optimise the OXGENE AAV and lentiviral packaging plasmids. This creates custom gene of interest (GOI) or Cap gene vectors at research grade from the new facility in Oxford, UK, which will also stock off-the-shelf packaging plasmids at research grade for rapid fulfilment.

GMP Grade AAV and Lentiviral Plasmid Manufacture and Supply

WuXi Advanced Therapies manufacture and supply OXGENE’s AAV and lentiviral plasmids at clinical and commercial GMP grades. They first establish research, master, and working cell banks under GMP conditions, then carry out stability studies and confirm the downstream processes and analytical assays to be used for quality control. GMP plasmid production runs are flexible in scale from 5-300L, and testing is fully integrated to ensure high purity and homogeneity and low endotoxin levels in the final product. WuXi Advanced Therapies’ manufacturing processes are antibiotic-free from clone selection to bacterial banking, a feature preferred by regulators for reducing the spread of antibiotic resistance and the risk of adverse reactions.

WuXi Advanced Therapies provides all the documentation required for investigational new drug (IND) filings, and their facility and quality management system has been designed according to FDA (USA), EMA (Europe), and NMPA (China) GMP compliance requirements.

To alleviate the industry-wide constraint around plasmid supply, and ensure rapid delivery worldwide, WuXi Advanced Therapies stocks OXGENE’s off-the-shelf AAV and lentiviral packaging plasmids at all grades. Even clinical GMP grade bespoke plasmids can be delivered within six months, with shorter timescales for research grade and clinical GMP plasmids.

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