Clarification and sterile Filtration of Antibodies Fast Cell Harvesting Without Using a Centrifuge
Whether employed in basic research or the production of biopharmaceuticals, monoclonal antibodies are used in an exceptionally wide range of applications. Until now, harvesting of mammalian cell cultures used to be time-consuming, especially at laboratory scale, and usually involved a centrifugation step. A new diatomaceous earth-based filtration solution now improves and accelerates this process significantly.
Monoclonal antibodies (mABs) are used in a wide range of applications including biopharmaceutical development, basic research and in vitro diagnostics. Expression systems of monoclonal antibodies typically utilise a signal peptide to ensure secretion of the antibody into the cell culture media.
Although this reduces the complexity of purification and avoids the need for cell disruption, it does require the use of expensive and/or time-consuming techniques to separate cells from antibody-containing cell culture fluid. This generally concerns the production of antibodies from mammalian cells, whether it be standard production using hybridomas or recombinant production in Chinese Hamster Ovary (CHO) or Human Embryonic Kidney (HEK) cell lines. At bioreactor scale, typically above 50 liters, this is usually done by processes such as continuous centrifugation and depth filtration [1, 2].
However, at research scale, which is typically less than 10 litres of cell culture per antibody, these processes become both impractical and expensive. The most widely cited method for research scale clarification is an initial centrifugation step followed by filtration of the cleared supernatant through a 0.2, 0.22 or 0.45 µm filter, either using a hand-operated syringe or a vacuum driven bottle-top filter. These are inexpensive and easy to use along with standard equipment present in most biotechnology laboratories. However, due to the shallow design of these filters, they are prone to clogging due to the presence of submicron particles remaining in suspension .
At Absolute Antibody, a British company specializing in expression, sequencing and engineering of antibodies, recombinant antibodies are transiently expressed in HEK293 and CHO-K1 cells. Their company lab facility typically works with approximately 50 antibodies per week from a scale of 30 mL up to 20 litres with a total weekly capacity of approximately 100 litres of cells. For the last six years, laboratory specialists have been using centrifugation followed by bottle-top filtration for all antibodies. As capacity at Absolute Antibody has grown, so has the need for an increased number of centrifuges. Over this time, lab engineers looked at a number of alternative options, including the use of filters designed for home brewing and flocculants such as Chitosan . These approaches proved to be slow, prone to clogging and low throughput and to be of low quality (e.g. endotoxin contaminated).
The Sartoclear Dynamics Lab V filtration system (Sartorius AG, Göttingen, Germany) was designed to reduce the time and effort involved in clarifying mammalian cell cultures. The addition of diatomaceous earth (DE) to cultures supports the formation of a porous filter cake to prevent blockage of the filter, allowing rapid removal of cells and cell debris from the sample (see Figure 1). This avoids the need for a centrifugation step, circumventing issues around centrifuge capacity and availability as well as preventing filters from clogging.
In the following, the Sartoclear Dynamics Lab V filtration system was tested and compared with the company’s standard process.
Materials and Methods
Suspension adapted HEK293 or CHO cells are grown in serum-free media and transfected with DNA plasmids encoding the heavy chain and light chain of a monoclonal antibody. The cells are harvested for purification 6 to 14 days post-transfection. For a typical expression batch of 1 litre, the original clarification process would involve centrifugation at 3,500 g in a bench-top centrifuge (2 × 500 mL) for 45 minutes followed by filtration using one or more vacuum-driven PES 0.45 µm bottle-top filters.
Filtered supernatant is then loaded onto an ÄKTA purifier with a 5 mL Protein A column for antibody capture and elution on low pH buffer. Antibody is then neutralised and proceeds either to additional purification steps (e.g. cation exchange or size exclusion chromatography) or directly to quality control depending on the requirements for the particular antibody batch. Quality control is performed by SDS-PAGE under non-reducing and reducing conditions, SEC-HPLC, endotoxin testing and, where required, an ELISA run to measure binding activity (see
In the modified downstream process, the centrifugation and filtration steps are replaced with the use of Sartoclear Dynamics Lab V. In the case of a 1-litre culture, 20 g of DE filter aid were added to 1 litre of cells. The cells and DE are mixed vigorously and then added directly to a 1,000 mL PES 0.22 µm Sartolab RF vacuum-driven filter included in the Sartoclear Dynamics Lab kit. A vacuum is applied and the clarified cell culture fluid collected in the 1 litre bottle. The filtrate is then taken through the purification and QC process as described above.