Manufacturing of Viral Vectors for Gene Therapy: Part I
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Pharmaceutical Review For reprint orders, please contact [email protected] 2 Review Manufacturing of viral vectors for gene therapy: part I. Upstream processing Pharm. Bioprocess. Manufacturing technologies for the production of clinical grade viral vectors have Otto-Wilhelm Merten*,1, been significantly improved in recent years. This is of utmost importance for gene Matthias Schweizer2, therapy approaches used in the treatment of inherited or acquired diseases. This Parminder Chahal3 4 article briefly describes the general principles for the production of viral vectors. The & Amine A Kamen 1Généthon, 1, Rue de l’Internationale, specific sections are dedicated to more detailed descriptions of the production of BP60, 91002 Evry Cedex 2, France adenoviral, AAV, γ-retroviral and lentiviral vectors. A subsequent article (the second 2Paul-Ehrlich-Institut, Paul-Ehrlich-Str. part) will then deal with downstream processing (purification) of viral vectors. 51–59, 63225 Langen, Germany 3Research Officer, Human Health Therapeutics Portfolio, National Over the last decade, manufacturing tech- produced as well as of the cell system used, Research Council Canada, 6100 Avenue nologies for the production of clinical grade different culture systems are available for Royalmount, Montréal, QC, H4P 2R2, viral vectors have been significantly improved. the production of viral vectors. With respect Canada This is of utmost importance for gene therapy to cell culture processes the following char- 4Bioengineering Department, Macdonald approaches used in the treatment of inherited acteristics are of importance and are briefly Engineering Building 270, McGill University, 817 Sherbrooke Street West, or acquired diseases. Up to January 2014, more described: constitutive versus induced vec- Montreal QC, H3A 0C3, Canada than half of all gene therapy clinical trials have tor production, culture of adherently grow- *Author for correspondence: made use of adenoviral, adeno-associated viral ing cells versus suspension cultures, multiple [email protected] (AAV), lentiviral or retroviral vectors [1] . Cur- process (parallel processing units) versus rently, there is a clear trend in an increased use unit process culture systems, and the use of of AAV and lentiviral vectors for gene delivery, serum-free versus serum-containing media. respectively, in vivo and ex vivo. In October 2012, the first European marketing authori- Mode of vector production zation for a human gene therapy product was In general, there are two modes of vector granted by the European Commission for productions: stable producer cell lines and Glybera®, which contains an AAV1 vector for transient (inducible) production system. 2 treatment of patients with lipoprotein lipase The induction of the vector production is deficiency[2] . performed either by transfection where the cells are transfected with one or several plas- Viral vector production mids providing the helper functions as well 2014 Viral vectors are derived from viruses that as the vector construct or by infection of the naturally infect human cells or other mam- cells with one or several virus(es) providing malian cells, thus their production is essen- the functions required for the production of tially using animal cell cultures and for some the viral vector. In principle, the transient systems, insect cell cultures. Therefore, ani- (or inducible) production mode can be used mal cell culture processes, involving cell cul- for the manufacture of all the viral vectors, ture bioreactors are needed for the industrial including retroviral vectors when stable production of viral vectors. In the follow- producer cell lines are not available. ing, cell culture technologies and processes are described, focusing on their use for the Adherent cell versus suspension cell specific production of viral vectors. Depend- In general, all cell lines except those derived ing on the amount of the viral vector to be from the blood system as well as tumor cells part of 10.2217/PBP.14.16 © 2014 Future Science Ltd Pharm. Bioprocess. (2014) 2(2), 183–203 ISSN 2048-9145 183 Review Merten, Schweizer, Chahal & Kamen and some insect derived cell lines grow adherently. Since • Low operation costs as compared with the multiple tumor cells have an increased tendency to grow in sus- parallel processes; pension they can be adapted to suspension growth [3]. The key difference between anchorage dependent and • Better monitored and controlled operations to suspension cells from a process standpoint is the way of insure optimal culture conditions. subculturing or passaging the cells. This can be as sim- In addition, multiple process systems (T-flasks, ple as dilution of the suspension cells in fresh medium roller bottles, CFs) are labor intensive, characterized or as complicated as detaching the anchorage dependent by an elevated contamination risk due to the need to cells from a surface and plating them on a new sur- open and close flasks for seeding, medium change, and face in fresh medium. Different ways to detach surface harvesting and can be used only in a batch, fed batch adherent cells for subcultivation have been reviewed by or repeated batch mode, whereas bioreactor systems Merten [4]. With regard to process scale up these differ- (stirred tank reactor for suspension and microcarrier ences become very important. For suspension cultures cultures, fixed bed, fluidized bed) can also be used in the size of the cell culture vessel can easily be scaled a perfusion mode – an option attractive for the pro- up from a spinner or shaker flask to a laboratory-scale duction of γ-retroviral and lentiviral vectors. Whereas stirred tank reactor (STR) then to an industrial scale fixed bed reactor systems show mass transfer limita- STR that might have a total volume of up to 30,000 l tions, hydrodynamic shear effects are the drawbacks of [5], whereas, for adherently growing cells, the scale-up suspension and microcarrier cultures. is limited by the surface available for the cell growth. In some cases, anchorage dependent cells have been Currently used cell culture systems for the adapted to suspension culture addressing this limitation. production of different viral vectors Another option largely adopted by the vaccine industry Small scale/laboratory scale cell culture systems to culture anchorage dependent cells is the use of micro- Adherent cells are cultured in static cell culture sys- carriers [6], which provides large surface areas in large tems including Petri dishes and T-flasks. T-flasks pro- volume bioreactors. Currently, virus production has vide culture surface ranging from 25 to 225 cm2. The been scaled up to 6000 l using microcarriers [7]. How- general applications are subculture, generation of seed ever, since the required vector quantities might be low material for starting larger scale productions as well as for some applications, viral vector manufacturers have small-scale productions of viral vectors, using batch, evaluated either fixed bed reactors or parallel processing fed batch or repeated batch culture modes. units, such as roller bottles or CellFactories (CFs). When larger viral vector quantities are needed for research or development purposes, anchorage depen- Unit process versus multiple parallel processes dent cells can be cultured in roller bottles (Figure 1A) Finally, cell culture systems can be distinguished by or multilayer tissue culture flasks such as CFs or Cell- ® their scalability. Unit process systems [8,9] are charac- STACKS (Figure 1B). Roller bottles provide cul- terized by scalability based on the increase in the size/ ture surfaces in the range of 490 cm2, 850 cm2 and volume of the culture system (‘bioreactor’) whereas 1750 cm 2, whereas CFs can provide up to 25,100 cm2 multiple culture systems [10] are characterized by scal- for 40 stacks. ability via the increase in the number of identical Roller bottles are simpler and easier to use than CFs culture systems (e.g., increase in the number of roller but require a mechanical device for rotation of the bottles or CFs for producing larger quantities of a given roller bottles at a maximum speed of 1.5–2 rotations/ product). min. The volume of the culture can be varied between In addition to the excellent scalability, further 25 and 100%, which is equivalent to 125 and 500 ml advantages of the unit process systems over the mul- for a roller bottle with a surface area of 850 cm2, thus tiple parallel processes are: allowing higher product concentrations at lower cul- ture volumes. When large numbers of roller bottles • Practicable to monitor and control a range of have to be used, then two solutions can be envisaged: process parameters with the use of detectors and control loops; • The use of automatic Cellmate processing systems as proposed by The Automation Partnership [102], a • Easy to generate and keep records of process system which allows the automatic handling of all conditions, enabling a better understanding of cell culture steps; such systems have been used by the process, which often results in improved cell the biopharmaceutical industry for the production growth and higher yields and provides extensive of viral vaccines for human and veterinary use as documentation for regulatory agencies; well as recombinant proteins; 184 Pharm. Bioprocess. (2014) 2(2) future science group Manufacturing of viral vectors for gene therapy: part I. Upstream processing Review • The use of the RollerCell system from Cellon [103], which is a self-contained roller bottle processing system. This system is designed to automate all steps of roller bottle based tissue culture – from cell inoculation, incubation, medium change and tryp- sinisation to final harvest. A single unit can process the equivalent of 200 standard roller bottles simul- taneously. A standard pack (20 bottles) provides a culture surface of 36,000 or 85,000 cm2 when using flat wall or expanded wall surface (pleated roller bottles), respectively. The same system in configuration of the ‘RollerCell’ 40 or ‘RC 40’ provides a culture surface of 2 × 36,000 cm2. On the other hand, the CF system is a static cul- ture system, mainly dedicated to the culture of adher- ently growing cells.