Application of ATR-FTIR imaging to industrial scale production of therapeutic antibodies

Biopharmaceuticals, proteins used as drugs, are an emerging area in the treatment of a range of diseases. The large scale production of these molecules requires a number of discrete steps including recombinant expression, isolation by a technique called column chromatography and development of an optimal final formulation. The isolation of the biopharmaceuticals from all other contaminating material usually involves a number of steps. The column chromatography used exploits a specific interaction between the target protein and another molecule immobilized on a support, the resin. Ideally all the contaminating material is washed from the column and the conditions are changed in order to release the protein from the resin. Optimal binding, washing and release steps often require significant changes in the properties of the surrounding solution, for example pH and ionic strength. In addition, it is possible to bind very high concentrations of the protein onto these resins. The concentrations reached are likely to be higher than those achieved at any other point in the production process. It is not known exactly what effects the different solution conditions and protein concentrations have on the quality of the protein. Indeed they may result in highly undesirable effects such as non-specific aggregation. Another key issue that scientists isolating biopharmaceuticals encounter is the binding of unwanted contaminant materials which reduces the ability of the column to isolate the target protein. Regular replacement of the resin significantly increases the cost of the isolation process and thus of biopharmaceutical production. Here we aim to investigate these issues with a view to improving isolation protocols using a specialized technique called Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. This technique allows us to chemically image (take chemical photographs of) the target protein molecules under different conditions both in static droplets and in dynamic systems similar to those used for large scale isolation. We can also directly image used resin with a view to understanding what contaminating molecules reduce the useful lifetime of the material. We anticipate that the results of these experiments will inform optimization of isolation protocols to both minimize target protein losses on-column and increase resin lifetime.

Biopharmaceuticals, proteins in particular antibodies, used as drugs are an emerging area in the treatment of a range of diseases. Large scale production of therapeutic antibodies requires a number of discrete steps including recombinant expression, isolation by column chromatography and development of an optimal final formulation. Previous and current successful BRIC grant proposals have addressed the issue of screening for protein quality in the final protein solutions. However one of the key unknowns in a bioprocessing pipeline is how do therapeutic antibodies behave in contact with chromatographic fixed bed adsorbers. Here we aim to use cutting edge Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopic imaging methods developed at Imperial College to investigate on-column protein behaviour and resin lifetime. ATR-FTIR can differentiate between protein, lipid and DNA molecules due to their specific vibrations. Thus we will directly assess what contribution these have to reducing resin lifetime by comparing resins with different levels of use. Using ATR-FTIR chemical imaging of antibodies in high-throughput droplets, as well as dynamic imaging on adsorbent resin and in microfluidic channels we aim to gain a greater understanding of the effects of the isolation process on protein behavior. We anticipate that the results of these experiments will inform optimization of isolation protocols to both minimize target protein losses on-column and increase resin lifetime.

The proposed research will provide novel insights into the behaviour of mAbs and scFvs on column aiming to shine a light on the least explored part of the whole bioprocessing pipeline, the protein isolation step. The methodologies and data generated by the research outlined in the proposal will be of significant interest and relevance to industry. We will engage with the BRIC industrial members at the regular dissemination meetings and in follow ups as appropriate in order to ensure that we are developing and investigating the correct model systems. In addition we hope to directly assess spent resin samples available from industry with a view to providing detailed feedback on the causes of reduced resin lifetime and to facilitate development of the ATR-FTIR chemical imaging methodology. We anticipate that the results of our studies will lead to applications for additional funding, for example CASE studentships or industrial partnership awards to further develop our research activities in the area of Bioprocessing.

The research described in the proposal describes novel applications of a cutting edge technique largely developed at Imperial College. It is anticipated that the PDRA employed to work on this project would gain significant expertise in protein chemistry and protein isolation as well as unique experience in the application of ATR-FTIR chemical imaging to understanding a relatively neglected area of the bioprocessing pipeline. They will also have regular contact with other Imperial based groups working in the area of bioprocessing in addition to building networks across the whole of BRIC. They will have ample opportunity to present their research findings at external meetings and will obtain significant training in writing scientific reports and manuscripts. It is also anticipated that they, along with the applicants, will be involved in the public dissemination of the research findings at a range of scientific outreach events.

A greater understanding of the effects of isolation of therapeutic molecules using fixed bed adsorbers on both the isolated protein and the resin will facilitate optimization of the isolation protocols to both increase yield of high quality material and increase resin lifetime. In the long term this should increase the efficiency of the isolation process and reduce the associated production costs. In addition, the research will also demonstrate the potential of ATR-FTIR chemical imaging as a diagnostic tool to determine the precise causes of resin fouling. Such an application may be of great benefit to industry when developing and marketing new products.