sign in sign up
<<
Home , Gene therapy

Article Reprint MAY 17, 2021

Ask the Expert – Best Practices for AAV Experts: Ann Rossi, Ph.D. and Cassie-Marie Peigné, Ph.D.

The majority of vectors used for gene are ● Transferring an optimized process at small currently generated using transient production scale into larger scale production vessels. systems, primarily due to convenience and speed of process. Manufacturing viral vectors via transfection ● Maintaining actively dividing cells and is fexible and efcient and does not require time culture health for efcient . consuming development of stable cell lines. However, transfection based methods do require Since the need for optimized transfection is so optimization to ensure the highest productivity, critical to successful AAV production, we felt it was consistency between batches, and scalability. important to provide the opportunity to ask questions about best practices in these areas. We have invited Key areas for improvement of transfection in AAV Ann Rossi Bilodeau, Ph.D., Senior Bioprocess production include: Applications Scientist, Corning Life Sciences and Cassie-Marie Peigné, PhD, Scientifc Support ● Identifying and addressing common Specialist, Polyplus-transfection to provide answers bottlenecks to scalable transfections. to reader questions on transfection for AAV production in applications. ● Streamlining the scale-up and transfection process.

● Designing and standardizing the process for consistent, reproducible results.

[email protected] CellCultureDish.com Reprinted with permission from Dish. The article originally appeared on CellCultureDish.com on 05/17/2021 How long does it take to see expression I’m looking for the easiest way to of AAV in HEK cells post transfection? determine the right AAV serotype for my Transient expression of AAVs can be observed in system. I’ve narrowed it down using HEK-293 cells as early as 24 hours post-transfection literature searches but is there an easy when using PolyPlus transfection reagents dedicated way to test a few serotypes. to production. The standard harvesting time is generally of 48-72 hours post-transfection, A deep dive into the literature is one of the best way irrespective of whether you are using adherent or to narrow down the list of potential AAV serotypes for suspension adapted HEK-293 cells. Depending on the different systems. Test the few top candidates, frst serotype, and on the gene of interest that the AAV on a small scale with some type of reporter construct vector carries, it can be worth harvesting at a later for an easily identifable to check for the time point, up to 10 days post-transfection or more. efciency of . There are commercially We would recommend harvesting at different time available testing kits that contain pre-made viral points to evaluate best productivity vs. stocks of multiple serotypes for GFP expression (e.g., cost-effectiveness of the process. Vector Biolabs’ AAV Serotypes Testing Kit AAV, GeneCopoeia’s AAVPrime™ Adeno-associated What is the best way to reduce the use of (AAV) Serotype Testing Kit). Finally, choose the one and/or keep transfection costs or two of the best from those tests and move onto testing with your gene-of-interest on a small scale. down? Indeed plasmid DNA is usually what brings up the Do you recommend sequencing plasmid cost of the transfection step. Less at bench scale, but before using? it quickly becomes expensive at larger scale, and Yes, we recommend sequencing before even more so when you need to start producing using use. Confrming the sequence and identify of GMP grade raw materials. To bring down the amount plasmids could ultimately save time and resources of plasmid used, the strategy is to primarily rely on a spent troubleshooting a transfection process that transfection reagent that can allow a signifcant might not ever yield infectious viral particles. reduction in plasmid DNA. Typically with PolyPlus transfection reagents, you can reduce the amount of Troubleshooting questions – what is the DNA by at least 20% compared to using alternative methods. In a second step, optimization of packaging limit for AAV and what transfection parameters, DNA/reagent ratio, VCD at happens if you get close to the limit? transfection and complexation time can further The size limit of AAV vectors is approximately 5 kb. improve productivity and therefore contribute to Some serotypes may accept larger , but most further decrease overall cost per AAV production literature cites success in packaging approximately batch. To take it a step further, if you have the 4.4 to 4.7 kb. Several publications have investigated fexibility in setting up your production system, the the effects of exceeding the AAV payload (e.g., HEK-293 cell lineage, the production medium are Grieger and Samulski in J Virol 2005, doi: parameters worth assessing to develop a process 10.1128/JVI.79.15.9933-9944.2005 and Wu et al. in that will be scalable to meet your AAV manufacturing Mol Ther 2010, doi: 10.1038/mt.2009.255). needs. In general, approaching the limit will affect the packaging efciency: the closer to the size limit, the lower the titer. However, there are many published reports describing alternative strategies for packaging larger , such as split AAV vectors and fragment assembly.

[email protected] CellCultureDish.com Reprinted with permission from Cell Culture Dish. The article originally appeared on CellCultureDish.com on 12/09/2020 In your experience, do you see a Both Corning and PolyPlus have Scientifc Support signifcant difference in AAV yield based Teams available to answer questions. on confuence and what do you ● Corning Life Sciences Scientifc Support recommend for confuence before ● PolyPlus Scientifc Support transfection? We are seeing inconsistent yields batch Cell confuence is a critical parameter for several to batch during transfection in a reasons. It has a direct impact on transfection efciency, and on the reproducibility and robustness . Do you have some of the process. It is important to refer to the troubleshooting tips? transfection reagent protocol, as the optimal Batch to batch variation can be addressed by confuence may differ when comparing several optimizing the transfection step in order to have a transfection reagents. robust production process. Parameters that are critical for the transfection step are the transfection For AAV production in adherent HEK-293 cells, we reagent used and whether the transfection reagent recommend using PEIpro transfection reagent. When can form every time the right sized complexes to using this reagent a confuency of 60-80% at the time effectively deliver the DNA cargo into cells. The of transfection is optimal because cells are still in preparation of the transfection mix is therefore key to their exponential growth phase. To improve achieve the right sized complexes. Key parameters to robustness of the process, think in terms of VCD at consider are the amount of DNA per million of cells the time of transfection and amount of DNA per for transfection, the DNA to reagent ratio, the mixing million of cells to ensure optimal confuency. strategy and length and the stability of the I’m looking to speed my AAV production complexes. With Polyplus transfection reagents, these parameters have already been optimized to and was wondering if you have tips or do simplify your task. They can be further refned or you recommend a production kit? adapted to your set up by contacting the Polyplus Production kits usually are expected to contain cells, Scientifc Support team. Lastly, it is important to medium and a transfection reagent. These are well make sure that the VCD at transfection remains the appreciated systems for bench scale production. same from one batch to another. However, using a kit poses two main concerns: 1) the lack of fexibility, and 2) the unsuitability for large We are looking for a good scale down scale manufacturing. model for our rAAV production, but have had trouble fnding one that is designed Whether at small or large scale, it is an advantage to be able to optimize production depending on the AAV for AAV production. serotype needed with the choice of the cells, the A good scale down model should mimic the AAV production medium and the transfection reagent. For production process at the fnal production scale, a production process to be scalable, it needs to be whether the fnal scale is intermediate or large, cost-effective, and usually complete production kits adherent or suspension. Though it may not be are not suited for large scale production and hence possible to miniaturize every aspect of upstream do not bring the beneft of cost reduction. It’s production, it is especially important for the model to important to have a screening step at small scale to achieve comparable cell expansion parameters and determine the best combination of tools (cells, media utilize a transfection process that is scalable. and transfection reagents) for maximum production Accordingly, choosing the right vessels for cell of full particles. An efcient way to ensure you are set scale-up/out and the appropriate transfection up for success is to contact scientifc support teams reagents are key to easing the transition from from the suppliers you are working with to ensure you process development to manufacturing. As an select optimal tools and have a streamlined process. example for adherent cultures: Corning® [email protected] CellCultureDish.com Reprinted with permission from Cell Culture Dish. The article originally appeared on CellCultureDish.com on 12/09/2020 HYPERFlask® or Corning HYPERStack® 12 layer improve delivery efciency and gene vessels can serve as the process development vessel expression. for a fnal scaled-out production in HYPERStack-36 vessels; Corning CellSTACK® 1 layer vessel or ● Cells and medium-choosing the best cell CellSTACK 2 Culture Chambers model larger scale culture system for highest production yield for cultures in CellSTACK-10 or CellSTACK-40 Chambers; the scale that you aim for, adherent or Corning CellCube® 10 Layer or CellCube-25 Layer suspension-based. Modules directly scale to CellCube-100 Layer Modules. We had a problem with contamination of our plasmid. What kind of screening or For suspension cultures, many bioreactor models are available in a wide-range of volumes for small removal do you recommend for through large scale cell culture. Once the cell culture endotoxin? platform is established, develop the transfection There are two basic options for low endotoxin process with reagents that are specifcally optimized plasmid preparations: perform your own preparations for direct scalability. and endotoxin testing or contract the preparations to an outside company. The decision which option is Polyplus Transfections’ PEIpro and FectorVIR are best comes down to weighing resources versus designed for robust and reproducible transfections in efciency and is highly dependent upon your adherent and suspension cells, respectively, to available lab resources, timeline, scale and fnal achieve high titer AAV yields from small scale application. For in-house preparation, utilize a process development through large scale commercially available plasmid prep kit that specifes clinical-grade manufacturing. low endotoxin or endotoxin-free plasmid preparation. Two questions, what do you consider Plasmid preparations can then be tested with a commercially available LAL (limulus amebocyte typical titer for AAV production and what lysate) assay kit. Alternatively, send your plasmid(s) can you do to increase it? to a company with plasmid production services, such as Aldevron that offers plasmid manufacturing for Typically, AAV production titers are around 10e11 to both research grade, GMP-source and GMP plasmids. 10e12 in VG/ml and 10e8 to 10e9 TU/ml. To put these numbers into perspective, it’s important to keep in mind that two AAV serotypes will most likely not As I am scaling up our adherent process, give the same yield, even when using the same I am not seeing titers scale at the same production process. AAV production yields vary ratio as in small scale. What can I do to depending on the serotype and the gene of interest. improve this? The best way to increase production of a given AAV is to go over critical parameters that directly have an First and foremost, think in terms of viable cell impact on the yield: plasmid DNA, transfection density rather than absolute surface area when reagent, cells and medium. optimizing a transfection process. In other words, calculate the amount of DNA and transfection ● Plasmid DNA- using a DOE approach, reagent per million of cells. Be sure to characterize /packaging/helper plasmid ratios, how the cultures grow (e.g., cell yield, doubling time) as well as the fnal DNA amount per million in the scaled-up vessel where you are conducting the cells can be refned to improve yield. transfection for an accurate assessment of cell density on the day of transfection. If possible, seed a ● Transfection reagent – comparing control vessel with the same growth kinetics to transfection reagents to reach highest harvest on the day of transfection to determine cell production yield, and further refning the density. Normalizing for cell growth in this way can plasmid DNA/ transfection reagent ratio to account for inconsistent cell growth between different sizes and/or types of cell culture vessels [email protected] CellCultureDish.com Reprinted with permission from Cell Culture Dish. The article originally appeared on CellCultureDish.com on 12/09/2020 and ensures reproducible transfections upon What are the biggest differences scaling-up. It is equally important that the between PEI and other methods for AAV transfection reagents are designed for scalable transfections, like those from Polyplus Transfections, transfection? Are there different media that enable robust transfection complex formation requirements? from small scale-process development through There are several transfection methods to produce intermediate to large scale production. AAVs. You may have come across more frequently -based transfection reagents such as PEI, There is a problem with plasmid access lipid-based and Calcium phosphate. What makes PEI and it is creating a real bottleneck in our and in general polymer-based transfection reagents process. Do you have any suggestions? more suitable to produce AAVs compared to lipid and calcium-based is the productivity and The best way to alleviate the bottleneck is to align cost-effectiveness. There are several PEI that are with a supplier that is capable of manufacturing commercially available, but only one PEI that is large-scale GMP-source or GMP plasmids. optimized for viral vector production: PEIpro®. We are weighing outsourcing vs. Compared to calcium phosphate, PEI-based transfection can decrease down by 10 times DNA producing our rAAV in house. Could you amount and can be used to transfect cells grown in talk about thoughts for each. the presence of or in the absence of serum. It brings There are several factors to consider when weighing versatility and scalability by being the go-to whether to outsource or produce rAAV transfection reagent compatible with most in-house—almost too many to expand on in detail in production platforms, adherent or suspension for this forum. Importantly, all steps from plasmid small to large scale production capacities. Compared generation to upstream cell culture and transfection to lipid-based transfection reagent, PEI-based through downstream purifcation and analytics must transfection is designed for large scale production: it be factored into the decision. Some of the major is cost-effective by substantially bringing down the factors to consider include: project timelines, the cost per production batch and is compatible with volume/titer needed versus your capacity to produce most production medium and complexation media to (i.e., what scale can your facility handle?), short- or adapt to each manufacturer’s process. Last but not long-term needs, research-grade versus GMP, cost in least, the availability of the transfection reagent at both materials and labor, application for the viral higher quality grade, ideally GMP Pharma grade, to particles, and available expertise to design and move on to producing clinical batches is necessary, troubleshoot the production and analytics. In the end, and must be taken into account in the choice of a it comes down to a question of whether it is better to reagent as early as possible to guarantee efcient relinquish control of the process for the sake of transition from PD to manufacturing. efciency or it is necessary and worth the investment of time, money, and resources for your specifc Meet the Experts application to produce in-house. For one perspective, the following article discusses the rationale of one Dr. Ann Rossi Bilodeau graduated company behind establishing in-house from the University of Rochester manufacturing: Markarian J. “Finding Success in AAV School of and Dentistry Manufacturing”. BioPharm International with a Ph.D. in Pharmacology and 2021;34(3):34–37. received postdoctoral training at the University of Chicago. Ann began her career with Corning Life Sciences as the Applications Lab Manager where she utilized her strong academic and industry research experience to direct the activities of the Applications Laboratory. Currently, Dr. Rossi Bilodeau is the Senior [email protected] CellCultureDish.com Reprinted with permission from Cell Culture Dish. The article originally appeared on CellCultureDish.com on 12/09/2020 Bioprocess Applications Scientist, functioning as the technical lead for generating applications in support of Corning Life Sciences’ Bioprocess and Cell/Gene Therapy portfolios including Corning® CellCube® and Corning HYPERStack® platforms.

Cassie-Marie Peigné is a Scientifc Support Specialist at Polyplus-transfection® SA, the leading company that supports Gene and Cell therapy, biologics manufacturing and life research with innovative nucleic acid transfection solutions. Cassie completed her PhD in Immunology at the University of Nantes, followed by a postdoctoral position in Infectious Diseases at the Okinawa Institute of Science and Technology, before joining Polyplus.

[email protected] CellCultureDish.com Reprinted with permission from Cell Culture Dish. The article originally appeared on CellCultureDish.com on 12/09/2020