CERTIFICATION
We’ve been recognized for our commitment to excellence and safety throughout the years - and we’re proud to tell you all about it! From day 1, we were getting noticed by the industry’s movers-and-shakers, and this has only pushed us to move forward towards greater advancements. And of course, all of these awards are testament to the incredible work that our team does day in and day out.
In terms of accreditation in Bioprocessing equipment, the most recognised standard is the ASME Bioprocessing Equipment (BPE) certification program. This certificate details the requirements applicable for the design of equipment that is to be used in the manufacture of health or other high hygienic requirement industries.
This standardised accreditation program is unique and is the result of widespread industry requests for such a certification to be created. It incorporates what are the current best practices, so if a biopharma/food company obtains certification, they can achieve cheaper development and production costs through increasing efficiency while maximising the quality and safety of their pharmaceutical/food at the same time. The BPE cert indicate that the components bearing the mark have been manufactured and certified by an ASME BPE cert owner. We received this certificate back in 2008 when were first starting up and have continued to work in according to this program.
PURIFICATION
Using this process 75%-80% of rAAV can be purified from crude lysate. The proteins present in the cell lysate aggregates rAAV and causes changes in its buoyant density. This results in rAAV being distributed across the gradient. To prevent this change in buoyant density, iodixanol (15%) containing 1M NaCl. The salt content destabilizes ionic interaction between macromolecules. For the rest of the purification process high salt was excluded from the gradient, so that the virus would band under the iso-osmotic conditions, which allows direct loading into further chromatography steps.
To purify the rAAV further, HPLC with cation exchange column will be used. Our column of choice is UNO S1, this column is derivatized with negatively charged, strongly acidic SO3 groups. Biocompatible polyetherentherketone tubing and fittings were used to process the samples. Only samples of 5ml could be injected into the HPLC machine, therefore we are using auto-sampler to load multiple 5ml injections with a 5ml injection of mobile phase to wash out the columns before the next injection of the sample. The UNO S1 columns bed volume is 1.3ml and is pre-equilibrated with PBS-MK buffer. Our sample loading and extracting speed is 0.5ml/min. Columns were washed with PBS-MK buffer until the iodixanol-induced absorption was reduced to near background levels.
EXTRACTION
Lysis
The freeze-thaw protocol is used to lyse our CHO cells and release their Viral contents. We use take the cell culture bag and freeze the cells in bath of dry ice and ethanol. It is then progressively thawed at a temperature of 37°C. Using this method of lysis we can get the cells to increase in size, swell and then rupture. As the freezing takes place, ice crystals are formed that expand and then eventually retract during the thawing procedure. We repeat this process 4 times to ensure that all of our cells are completely ruptured. It is a time consuming process, but given the massive return on investment seen with Adenogen both in terms of money and product yield, it is a worthwhile endeavour
Filtration
The combination of the lysed cells and RAAV must be separated once the cells have been lysed. To accomplish this we employ a dead end filtration system. Through means of a constant pressure of 15 PSI to control flow rate speed, The system consists of a pipe like structure that has a filter mesh located perpendicular to the flow path of the combined RAAV and lysed CHO cells. This filter has a mesh of 10µm. This is a mesh that is large enough to allow the RAAV to pass through, and fine enough to prevent the considerably larger CHO cells from passing through. These CHO cells form a filter cake. As this is a single batch procedure, it is a disposable apparatus.
BIOREACTOR
As already stated we are using the AAV, we have genetically enhanced the AAV to carry a healthy and functional gene that will replace a mutated and faulty one that is causing a certain disease to be expressed in the body. In our case we are speaking about cancer. Seeing as this is a very technical idea the process needs to be precise and accurate. The first step after acquiring all the raw materials such as the mammalian cells, cell media and the AAV itself, is to set up the bioreactor. As we are using fed batch the vital nutrients will be added into the bioreactor with the mammalian cells and the genetically modified AAV. The temperature and pH will be set at 37°C and pH 7.1 respectively. The agitation speed will be set between 18rpm and 28rpm to ensure the cells are subjected to substantial mixing and gas transfer, the constant agitation also prevent the settling and allows for oxygenation. As lactic acid is a by-product of cellular metabolism it needs to be removed, this will be done using specially designed integrated extraction valves. When lactic acid does acid there in a rapid drop of pH, we will use this as an indicator to add in more cell media to continue the growth of the mammalian cells. Through our research we have decided to add in the media when we see a decrease in pH between > 0.1 - 0.2.
OUR PROCESS
Every project is taken very seriously by our team at CellTech and the design of our therapeutics manufacturing process was no exception. This process was designed with highest yield and top quality in mind. Our team uses most recent technologies to achieve the most efficient design of the process to manufacture rAAV.