With the advance of cell culture technology, higher and higher levels of protein expression have become a reality. For example, 10-20 gram / litre of antibody could be produced in mammalian cell broth in comparison to previously 1 gram / litre level. Protein A affinity chromatography is the standard method for the capturing of antibodies in the bioprocessing industry. Currently this affinity chromatography technology has almost reached to its physical capacity limit (e.g. 60-70 mg/ml resin). It won’t be cost-effective to purify such high-titer antibody (e.g. 10-30 g/ml titer) using Protein A media. In addition, the liquid-volume-reduction factor seen in the low-titer process will decease, i.e. the liquid volume post the Protein A step will be the same or even higher than the initial broth volume.

Alternative technologies such as crystallisation / precipitation, smart polymer adsorption and aqueous two-phase extract are being explored and evaluated in the industry. However, none of the above techniques shows superior performance and, incorporating such techniques to the current manufacturing process is hugely challenging in respect of the engineering compatibility and operational efficiency.

When the antibody is expressed in high titers (e.g. 10-20 mg/ml), the content of the impurities such as host cell proteins, host cell DNA, virus, pigments and other small nutrition molecules (e.g. growth factors and cholesterols) becomes the minority, i.e. much less than 50% in comparison to the content of the target antibody.

Instead of capturing a high-titer antibody with expensive affinity media of restrictive binding capacity, we propose to capture the impurities but let the pure target antibody passing through chromatography columns. It is called negative chromatography antibody purification (NCAP) technology. A group of mixed-mode ligand and ion-exchange chromatography media with complementary binding spectrums can be deployed to efficiently capture all the impurities. Such type of media is much stable and costs much less. There is no engineering restriction or modification required to adopt the NCAP to a company’s existing manufacturing facility. Accordingly, the capacity bottleneck facing the affinity medium could be resolved at a cost-effective way.

A few example scenarios deploying the NCAP are as follows:

The negative chromatography media could be deployed immediately after the cell culture step. A mixed-mode ligand medium can be mixed with the cell culture broth before the cell harvest step or, a depth filter cartridge filled with the mixed-mode resin is deployed in the recovery step.

The clarified antibody feedstock can be pumped through a serial of negative chromatography columns to get all the impurities removed. Ultrafiltration may be introduced in-between to reduce the liquid volume.

The NCAP technology could potentially show a perfect fit to the antibody precipitation process. The impurities that affect a given precipitation process could be effectively removed by the negative chromatography step in advance. The remaining impurities co-precipitated with the target antibody could be effectively removed by another negative chromatography step.

The NCAP technology can be readily incorporated into a batch or continuous purification process.

BioToolomics has developed the technology and the facility to produce high capacity low cost chromatography media coupling with ion-exchange and mixed mode functional groups. We have the large mixed-mode ligand library (MMLL) against all type of impurities.

By the combination of our MMLL, innovative coupling technology and low cost high porous resin technology, BioToolomics would like to offer this unique platform technology to biomanufacturing companies in developing their much efficient and cost-effective process.

BioToolomics takes the OMICS approach, through the collaboration with North East Proteomics Analysis Facility (NEPAF) in Newcastle upon Tyne UK, to map out the component profiles in the cell culture broth. The purification strategy to remove all the impurities is designed accordingly. Screening of the MMLL coupled to the resins (including other commercial resins) is then carried out against the impurity populations. The findings are fed back to modify the purification strategy. The chosen resins are subsequently made in small scale following with small scale process development work. The results are fed back to fine-tune the purification strategy. Development and scaling-up work is then conducted.

By adopting the NCAP technology platform, a biomanufacturing company can bring all the relevant personnel (such as upstream R&D, downstream R&D, analytical department, QA/QC and production department, regulatory staff) together to design a high-quality purification strategy / process even before any practical work starts. It could greatly reduce the overall development cost and time.

BioToolomics can provide the screening and process development service or work with biomanufacturing companies to develop new platform processes together. BioToolomics also has the capability to supply chromatography resins and columns at large quantity.

For more information or possible collaborations, please contact us.