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.

Protein A affinity medium is very costly and not very robust. It always imposes a huge inventory burden. Any kind of mishandling may mean a significant financial loss. The leaked protein ligand is another contaminant in the production stream and has to be removed by an extra chromatography step.

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

Instead of capturing the target antibody with expensive affinity media, we propose to capture the impurities but let the target antibody pass through. A group of ion-exchange and mixed-mode (multi-mode) ligand (MML) chromatography media with complementary binding spectrums can be deployed to efficiently capture all the impurities. This is called negative chromatography antibody purification (NCAP) technology. Such type of media is very caustic-stable and much cheaper than protein A resin. The MML media generally has a much higher binding capacity as well. By adopting the NCAP technology, very cost-effective purification processes could be developed.

BioToolomics has built up a large mixed-mode ligand library (MMLL) against all the possible impurities. BioToolomics takes the OMICS approach, in partnership with the North East Proteomics Analysis Facility (NEPAF) in Newcastle upon Tyne UK, to map out the impurity profiles surrounding a given antibody and to establish the 3D relationships (hydrophobicity, pI and size) of the impurities. Mixed-mode resins are selected and screened at various conditions to achieve the full removal of the impurities. BioToolomics will then produce chromatography resins for large-scale manufacturing applications. We guarantee the cost of such mixed-mode resins will be low enough for use in single batch operations, i.e. fresh resin will be used for every bioprocessing batch. There will be no need for re-use, re-cleaning and re-validation activities.

BioToolomics would like to offer this unique platform technology to biopharmaceutical companies to develop more efficient and cost-effective purification processes.

For more information or possible collaborations, please contact us.