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

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description 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 Reflection -Fourier Transform Infrared (ATR-FTIR) spectroscopic imaging. This technique allows us to chemically image (take chemical photographs of) the distribution of the target protein molecules under different conditions both in static droplets and in dynamic systems similar to those used for large-scale isolation.
Our first studies were a proof of principle that we can use this technique to study proteins under a range of conditions that would normally be experienced during the isolation process using purified protein samples in solution. The findings of this research have been recently published in Analytical Chemistry. In addition we have developed methodologies to allow us to study the changes in the chromatographic resin during cleaning processes key to extending the lifetime of the resin. Our ATR-FTIR spectroscopic methods allow us for the first time to explore changes in the chemistry of the resin under harsh cleaning protocols. Our results revealed that even under comparatively low concentrations of NaOH (0.1 M) the Protein A ligand on the resin undergoes significant changes in conformation. This change in conformation of the Protein A ligand is likely to contribute to reductions in efficiency of purification over time. Our analysis also showed that the addition of trehalose reduced the level of conformational change. Taken together these findings could help to optimise cleaning protocols and extend resin lifetime and reduce mAb production costs. These findings have been published in Analytical and Bioanalytical Chemistry.
Our latest research has built on earlier studies to develop the first in-column ATR-FTIR spectroscopic analysis of gel liquid chromatography. With this novel microchip based approach, we investigated the fouling process in situ during affinity purification. Our results on this part of the project were published in Scientific Reports in 2016. Additional work focused on attempting to develop an online ATR-FTIR system which allows real time measurement of the changes in proteins on a full-size purification column during a purification. We were partially successful in this prior to the end of the grant and aim to submit a proposal to continue this work later this year.
Exploitation Route We anticipate that the results of these experiments will inform optimization of isolation protocols used in industry to both minimize target protein losses on-column and increase resin lifetime. This will be of major interest to biopharmaceutical companies and should in the long term play a part in reducing the astronomical costs associated with development and production of therapeutic antibodies. We have an additional studentship, an iCASE, with Bristol Myers Squibb, who is building on the work that was carried out as part of this funding. We currently have a paper describing this under review at Analytical Chemistry. The student started in Octber 2017. We were also awarded BBSRC Follow-on-Fund application to develop our on-column analysis tools. Support letters for this application were written by PALL, MedImmune and GSK. As a result of our discussions with GSK for the Follow-on-Fund application we were awarded an additional GSK/BBSRC CASE award studentship to focus on the use of Raman spectroscopy for analysis of proteins on-column.
Sectors Education,Pharmaceuticals and Medical Biotechnology

 
Description The PDRA appointed, Dr Maxime Boulet-Audet, has been trained in a variety of protein production methods and has become familiar with the equipment set up for the ATR-FTIR measurements. He has additionally attended a course on spectroscopic methodologies and a week-long BRIC workshop where he was able to interact with both other academic scientists as well as experienced industrialists with an interest in industrial biotechnology. Dr Boulet has also been able to establish an internal collaboration which will significantly augment his research. We will continue to provide him with a range of training opportunities with a view to making him as employable as possible after completing this contract. Both Dr Byrne and Prof Kazarian have had the opportunity to present their research to the wider general public. Dr Byrne presented some of her research at the Ada Lovelace day in October 2013. Prof Kazarian has presented his research at the Imperial College Fringe. Dr Boulet has had the opportunity to present his work at a several BRIC events as well as at a high profile specialist spectroscopy meeting in the US. Dr Boulet has subsequently joined a small start up company in San Francisco working on using FTIR spectroscopic methods to characterise recombinant silk proteins for clothing manufacture. SInce our last submission Kazarian (Principal Supervisor) and Byrne (co-Supervisor) have been awarded an iCASE studentship in partnership with Bristol Myers Squibb to carry on the microfluidic studies started by Dr Boulet during his BRIC funded post-doc in our groups. The student started in Octber 2017. We have both also applied for and are currently awaiting the outcome of a BBSRC Follow-on-Fund application to develop our on-column analysis tools. Support letters for this application were written by PALL, MedImmune and GSK. As a result of our discussions with GSK for the Follow-on-Fund application we have been awarded an additional GSK/BBSRC CASE award studentship to focus on the use of Raman spectroscopy for analysis of proteins on-column. We have appointed an excellent person to this PhD position. We have additionally been awarded a Follow-on Fund grant to continue our research in this area and work has begun on this project. We have presented extensively on this work and were additionally asked to put together a specialist Protein Biotechnology session at the SciX2018 meeting in Atlanta on the basis of our expertise in this area.
First Year Of Impact 2014
Sector Education,Manufacturing, including Industrial Biotechology
 
Description BBSRC iCASE
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2021
 
Description On-Column Monitoring of Protein Purification by Spectroscopic Techniques
Amount £100,000 (GBP)
Funding ID BB/S506965/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2018 
End 09/2022
 
Description Towards on-column monitoring of protein purification by ATR-FTIR and Raman Spectroscopy
Amount £199,240 (GBP)
Funding ID BB/R019533/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2018 
End 11/2019
 
Title Use of ATR-FTIR to study antibody behaviour 
Description The method allows us to study in small scale changes in the behaviour of mABs in solution under conditions likely to be encountered on-column. The novel set up used allows us to monitor for changes in aggregation status. 
Type Of Material Antibody 
Provided To Others? No  
Impact The method has been published. 
 
Description Collaboration with GSK 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution We will supervise a BBSRC/GSK funded PhD student to continue aspects of the work started during the BRIC project
Collaborator Contribution They have provided the studentship funding and will also host the student for periods of time during the PhD.
Impact None as yet, the student only starts in Oct 2018.
Start Year 2018
 
Description Isolation of antibodies 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We are isolating the mAB sample from cell culture medium provided by Dr Kontoravdi's lab and as a result we are providing samples containing the contaminants produced during expression. These are being used by Dr Kontoravdi's lab.
Collaborator Contribution Dr Cleo Kontoravdi's group have provided us with mAB which they produce as a by-product of their research.
Impact The protein provided by Dr Kontoravdi was used in our published study (see other sections)
Start Year 2013
 
Description PALL antibodies 
Organisation PALL Europe
Country United Kingdom 
Sector Private 
PI Contribution PALL Europe have provided antibodies expressed in culture fluid for us to purify and analyse.
Collaborator Contribution We have used the antibodies for purification and fouling analysis of the purification resin.
Impact None yet
Start Year 2015
 
Description IMperial College Fringe 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Educated the public

Greater knowledge in the public
Year(s) Of Engagement Activity 2014
 
Description Imperial Fringe 2016 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The imperial Fringe is a 3 day event designed to showcase the broad portfolio of research activities to the general public. The public get teh opportunity to try out simple experiments and to discuss the science with researchers.
Year(s) Of Engagement Activity 2016