STORAGE
Purified rAAV can be stored up to 2-3 weeks at temperature of 4 degree Celsius, this can be achieved by simply placing the vials in most of the common fridges as long as the temperature requirement is met. Although even after storing it at 4 degrees Celsius for longer time period than 3 weeks, no significant loss in viral count has been recorded. It is still advisable and recommended to store the vials containing rAAV at -80 degrees Celsius if longer periods of storage are required. For this kind of long term storage, it is recommended to use VWR® Liquid Nitrogen Cryogenic Freezer Boxes with Drain Slots.
PACKAGING
The rAAV after the production would be stored in Duran glass vials with a container closure system caps. These caps provide a safe way of extracting the virus from the bottle into the syringe without any contact with the viral particles. The Duran vials would contain phosphate buffered saline (PBS) solution with 5% glycerol which is suitable for in vivo injections. Each vial containing enough of the viral particles for three injections. Adenogen comes in a box of ten vials or ten first-time treatments. Each of these is transported in liquid nitrogen which keeps them at an adequate temperature and ensures that they will not undergo degradation during transportation period.
PURIFICATION
Purification of rAAV is rather simple and the process we are using to purify the our recombinant AAV vectors can be done within one day. During this purification process, we will be using Iodixanol at 15% gradient, to cushion the rAAV particles. Iodixanol was originally produced as an X-ray contrast compounds, but it is used in our process due to its ability to maintain the solution in iso-osmotic condition at all densities. This property makes iodixanol a perfect solvent for our purification using HPLC machine. HPLC of choice for this process is Beckman System Gold HPLC hardware with UNO S1 cation exchange column will be used to further purify the our recombinant AAV. For final stage of purification BIOMAX 100 (milipore) dialysis would be carried out on the solution.
EXTRACTION
For the freeze thaw part of the operation we use the Celsius® FFT (3T, TPE) STD-S FZB212435.
It is a ready-to-use container, composed of a bag and a protective shell, designed for freezing and thawing. They come in a multitude of sizes but our operation in its current scale requires two per batch (2x12L)
Some key features and components of our system include:
•HDPE shell
•Sturdy
•Disposable
•Large supply available
•Bags are integrity tested
• Extensive safety features
•Standardized design
•We use Readymate™ adapters that are compatible with our Filtration system
For our Filration system we have a dead end filter operation. It is a disposable ULTA Prime CG Capsule Assembly made by GE healthcare. This system is connected to a pump valve and that provide the required pressure. The ULTA Prime CG 0.2 µm 2" capsule filter The assembly consists of C-Flex® 374 tubing and with connectors that are compatable with our frezzer bag apparatus.
Some key features and components of our sytem include:
•Gamma irradiated for ultimate sterility
•Single use Readyfilter™ assembly in the piping
•We use a 10µm filter but can go as low as .02µm if required
•Consists of Readymate™ adapters that are compatible with our Freeze thaw bags
•Due to compatible adapters, setup is extremely quick
•Due to disposable nature, no cleaning is required, lowering cost
ANCILLARIES
The PALL XRS 20 bioreactor system comes equip with acid/base pH control feed, an integrated single use pH and dissolved oxygen (DO) sensors, these are important to monitor the levels of pH in the bioreactor and the levels of (DO) respectively. Oxygen is provided using an air mixing controller providing a healthy and rich supply of oxygen to allow for mammalian growth to prosper. Carbon Dioxide is provided using an aeration and sensor system. There are samples ports located in appropriate positions to unsure easy usage, as already mentioned sampling can be carried out without stopping the process ensuring no time or money is wasted throughout the progression. The PALL XRS 20 bioreactor system is up to date with the latest technologies, containing touchscreen displays to simplify setup, calibration, operation, alarms, trending and process data collection. Furthermore, if we wish to perform benchtop operations, we have the properties to allow this. A filter heater comes with the PALL XRS 20 bioreactor to prevent water condensing, leading to clogging and overpressure in the bag. In addition, a load cell is integrated into the bioreactor system, this is important to monitor the growing weight to indicate when the process needs to be stopped, to ensure no time or money is wasted. The load cell will also be used to indicate when nutrients gases and other materials need to be added into the bioreactor to ensure healthy growth continues. Understanding when these need to be added is imperative and the load cell will be used to measure these times. Below is a short run down of the ancillaries that are integrated with our chosen bioreactor system.
•Powers intuitive graphical displays, including synoptic view, summary screen, trend graphing
•Enables automatic process control of agitation, temperature, pH, DO, gas flow and mixture
•Allows on-screen calibration for sensors and pumps
•Supports user definable P&I settings for fine tuning of process control loops
•Customizable alarms for process monitoring
•Customizable loop trending for up to 8 variables, simultaneously
•Stores up to 10 user-definable recipes Assigns peristaltic process pumps for additions and harvest
•CFR21 Part 11 compatible. Multilevel security to limit setpoint control access
BIOREACTOR
A wave bag is a relatively new type of bioreactor, designed just before the millennium as an alternative to the classic steel bioreactors. They are cheaper, requires no cleaning as the bags are disposed at the end of the process and no validation between batches is needed. Due to the wave bag being designed to incorporate a non-invasive rocking motion that allows for substantial mixing and gas transfer to provide optimal cell growth. This non-invasive rocking prevents settling and provides oxygenation without producing bubbles (Why is this important) As technology has advanced exponentially since the late 90’s, the modern wave bags come equipped with “fully integrated systems”, “intelligent control software” and “advanced sensor technology. The key aspect for the wave bag is the limited contamination, since the bag is disposed of and no cleaning is required contamination is of little concern. Furthermore, the sterilisation process is removed due to this aspect, reducing times and costs. The idea of scaling up is of course a goal for us, if the demand is met. From our research it will be possible to scale up the wave bag. A 580litre wave bag has been demonstrated for scientific usage.
As our process involves the adeno-associated virus, the type of bioreactor was imperative to allow optimal reproduction of both our cell line and the AAV virus. The wave bag bioreactor uses a closed system which is ideal for virus production. From research it has been estimated that with 293 mammalian cells that have been grown in suspension and then infected with a recombinant adenovirus. Cells grew to 4x106/ml and virus production was 100,000 virial particles/cell. Using this Wave bag bioreactor it is possible to produce viruses without the risk of contamination, removing the cause for a biosafety cabinet.
The bioreactor we shall be using is the PALL XRS 20 bioreactor system, it is designed for the cultivation of mammalian cells in suspension culture under controlled conditions. This is exactly what will be needed for our bioprocess. The bioreactor is comprised of three components (XRS 20 Platform, XRS 20 Controller and the Allegro™ XRS 20 biocontainers). We chose this bioreactor specifically as it has been shown to produce superior performances compared to other competitors on the market. This can be seen from the unique Bi-Axial agitation which produces shorter mixing times and higher mass transfer properties. Another important aspect with took into account when choosing the bioreactor system we will use was the type of fermentation we will use. The PALL XRS 20 bioreactor system is perfect for fed batch fermentation, the type we will use. It handles fed batch extremely well. There are of course many other advantages to the PALL XRS 20 bioreactor system including
•Fully enclosed platform with safety interlock
•Improved operator safety
•Suitable for production environment
•Protects light sensitive culture media
•Integrated filters
•Integrated optical sensors
•Inlet and exhaust filters
•Sample testing without stopping process
•Easy installation
CELL LINE
Chinese hamster ovary (CHO) cells are an epithelial cell line derived from the ovary of the Chinese hamster, often used in biological and medical research and commercially in the production of therapeutic proteins. They have found wide use in studies of genetics, toxicity screening, nutrition and gene expression, particularly to express recombinant proteins. CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics. This makes these cells our cell line of choice as it is perfect for the production of the AAV. CHOZN® DHFR-/- ZFN-Modified CHO Cell Line requires EX-CELL® 302 Serum-Free Medium for CHO Cells.
Growing CHO cells requires a lot of media supplements, these are as follows:
1.Albumin human recombinant, expressed in rice, lyophilized powder, cell culture tested, low endotoxin, ≥96%.
It is ideally suited for use in cell culture media for Chinese Hamster Ovary (CHO), hybridoma, VERO, stem cells and primary cells. It is safe and regulatory friendly. Eliminates animal derived additives.
2.Antioxidant Supplement
Proprietary medium supplement specially formulated to boost cell growth for enhanced viral and protein expression
3.Bioreactor pH Adjustment Solution
Concentrated carbonate/bicarbonate solution designed specifically to adjust pH in bioreactor applications. This buffered solution is used to adjust the pH in bioreactors, minimizes detrimental localized pH effects and the osmolality impact compared to using NaOH.
4.EX-CELL® Antifoam
EX-CELL® ANTIFOAM is a USP Grade non-ionic emulsion (simethicone) designed for use in the pharmaceutical and veterinary biological industries to aid in the control of foaming typically associated with the use of culture medium in bioreactors.
5.EX-CELL® Glycosylation Adjust (Gal +)
A protein quality supplement that targets glycosylation attributes, EX-CELL®Glycosylation Adjust (Gal+) allows to easily and quickly achieve desired N-linked glycosylation by increasing the galactose site occupancy on the oligosaccharide to a higher level.
6.Fatty Acid Supplement
Animal-component free. Proprietary aqueous mixture of fatty acids that have been found to be optimal for cell growth and protein expression. Recommended for use at 0.25 to 0.5 ml per liter with CHO cells.
7.GSEM Supplement
The glutamine synthetase (GS) system is used to express recombinant proteins from mammalian cells. One mL will supplement 50 mL of cell culture medium.
8.HyPep® 4601 Protein Hydrolysate from wheat gluten
Rich source of L-glutamine for medium supplementation at 1 to 5 g/L of medium. It also provides amino acids that support the general nutritional requirements of cells in culture.
9.LONG® R3 IGF-I human
LONG® R3 IGF-I is a recombinant analog of human insulin-like growth factor-I (IGF-I) that has been specifically engineered for the enhancement of cell culture performance. It is more biologically potent in vitro than either insulin or native IGF-I and has been shown to significantly increase recombinant protein production.
10.Polyamine Supplement
General medium supplement specially formulated to boost cell growth for enhanced viral and protein expression. Used in combination with Antioxidant Supplement.
The cell line, media and all its supplements are available from ”Sigma-Aldrich” suppliers. It is very convenient that all the raw materials are available from the same supplier, making the shipping and ordering process a lot simpler and easier to handle
ADENO - ASSOCIATED VIRUS
Adeno-associated virus (AAV) is a small virus which infects humans and some other primate species. AAV is not currently known to cause disease. The virus causes a very mild immune response, lending further support to its apparent lack of pathogenicity. Gene therapyvectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell, although in the native virus some integration of virally carried genes into the host genome does occur. These features make AAV a very attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models. AAV is a small (25-nm), nonenveloped virus that packages a linear single-stranded DNA genome. It belongs to the family Parvoviridae and is placed in the genus Dependovirus, because productive infection by AAV occurs only in the presence of a helper virus, either adenovirus or herpesvirus. In the absence of helper virus, AAV (serotype 2) can set up latency by integrating into chromosome 19q13.4, establishing itself as the only mammalian DNA virus known to be capable of site-specific integration. There are two stages to the AAV life cycle, after successful infection, a lytic stage and a lysogenic stage. In the presence of helper virus (adenovirus or herpesvirus), the lytic stage ensues. During this period, AAV undergoes productive infection characterized by genome replication, viral gene expression, and virion production. The adenoviral genes that provide helper functions regarding AAV gene expression have been identified and include E1a, E1b, E2a, E4, and VA RNA. Herpesvirus aids in AAV gene expression by providing viral DNA polymerase and helicase as well as the early functions necessary for HSV transcription. Although adenovirus and herpesvirus provide different sets of genes for helper function, they both regulate cellular gene expression, providing a permissive intracellular milieu for AAV productive infection. In the absence of adenovirus or herpesvirus, there is limited AAV replication, viral gene expression is repressed, and the AAV genome can establish latency by integrating into a 4-kb region on chromosome 19 (q13.4), termed AAVS1. The AAVS1 locus is near several muscle-specific genes, TNNT1 and TNNI3. The AAVS1 region itself is an upstream part of a recently described gene, MBS85. The exact function of this gene is not clear, but its product has been shown to be involved in actin organization. Whether AAV integration into this site is suitable for human gene therapy applications remains to be evaluated. Tissue culture experiments suggest that the AAVS1 locus is a safe integration site. The only reasonable limitation of AAV appears to be the fact that it only has a space for 4.7kb which means that the size of genes getting replaced would be limited. This is being worked on at the moment, by developing new ways of treatment using rAAV, and by developing new serotypes of rAAV. These new ways of treatment would be by using several serotypes of rAAV to cut and replace DNA at different locations, thus increasing the amount of genes being replaced.
At BIOplus CellTech, our manufacturing methods go hand-in-hand with the quality materials that we use. We aim to create the highest quality products that last, and we do that by utilizing grade-A raw materials that pass our vigourous inspections process. We’ve built our reputation on creating products that you can trust for their endurance. If you have any questions, please contact our customer service team today.