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AIR2-CT93-0911
The Development, Investigation and Assessment of A Novel Continuous Affinity Separations Technique |
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Contract No | AIR2-CT93-0911 |
| Total Cost | 1 347 816 | |
| EC Contribution | 673 908 | |
| Start Date | 01/11/1993 | |
| Duration | 36 months |
SUMMARY
This project involves the development of a process for separating pure proteins, such as enzymes or other products expressed by recombinant (genetically manipulated) organisms. The principle is based on the established procedure in which an anti-body which will react with the required protein is linked to a support (which may be packed in a column) producing an affinity material to which the sample material is brought into contact. The required protein binds onto the column, the impurities are washed off and the required protein is eluted in a pure form. This is a batch process. The purpose of this work is to replace this with a continuous method, binding the affinity material to a looped belt, which then passes through the crude sample, the washing process, a chamber where the required product is eluted and finally is regenerated. In spite of technical difficulties which initially limited yields and process rates, encouraging results were obtained, which should result in development of an effective apparatus.
INTRODUCTION
The most effective methods of protein purification involve the removal of the target protein from solution
by bringing it into contact with a solid that it can bind to. The binding between the solid and the protein
can be made very specific by attaching a compound to the solid that is known to bind very effectively
with the desired protein (affinity ligand). This is commonly carried out by column chromatography, where
the solid adsorbent particles are packed into a column. The crude mixture is passed through the column
and the target protein binds to the particles. After the contaminants are washed away, another liquid is
passed through the column that removes the attached protein from the particles, which is then recovered
in solution. In the novel continuous separation method developed here, the solid is a belt of material in a
continuous loop which moves through a series of chambers containing flows of the liquids necessary for
the purification. In contrast to the column format where each step takes place in sequence, in this method
all processes are occur simultaneously on different parts of the moving belt. The overall result is that a
constant flow of crude material can be processed and a constant flow of purified protein is produced. In
addition, since the rates of adsorption and desorption were not limited by the diffusion of the solute into
a matrix, faster ligand cycle times could be used. The potential advantages are lower costs through the
use of small amounts of affinity ligand, inexpensive support materials, and continuous operation.
ACTIVITIES
This project involved several areas of work that were integrated into the development of this affinity separation
method. Apparatus was designed and constructed, and this was tested with several different protein/ligand
affinity systems. Studies of the factors affecting separation performance provided insights into the limitations
of both the apparatus and the affinity systems that led to improvements in design and methods of operation.
APPARATUS
Three versions of apparatus were used during the project. Initially, basic apparatus was tested. This was later
replaced by 2 professionally constructed prototypes of increasing sophistication. The basic design consisted
of a multi chambered tank, a belt drive system, and pumps for the delivery of liquid media. The most advanced
prototype had built in pumps, drive and control electronics, and a multi roller drive mechanism.
For the majority of work, nylon belts were used that were activated by partial hydrolysis. Ligands were attached
via gluteraldehyde. This was not satisfactory for all separations due to non specific binding of proteins. A new
material was developed by Whatrnan Ltd. that may be better for some applications as it shows low levels of non
specific binding. Methods were devised that enabled this to be produced in a belt format that was chemically
robust enough for the ligand attachment process and physically strong enough for use in the continuous apparatus.
This was not tested in continuous separations as it was only available at the end of the project.
MODEL SYSTEMS
A number of target/ligand affinity systems were investigated on the principle that each could give unique information
on the effectiveness of the method. These were: trypsin/trypsin inhibitor, IgG/Protein A, histidine recombinant
protein/metal chelation, streptavidin binding recombinant protein/streptavidin, b-galactosidase/ inhibitor,
Cu2+/chelation.
RESULTS
The most successful of the experimental systems was that based on trypsin. This gave stable and reproducible
results and was therefore used for much of the testing and development work. Continuous purifications of trypsin
from pancreatic extract were carried out, the longest being of 30 h duration. The recovery was 30 40% at
approximately
0.6 mg h 1, and only one contaminant was detected in the product. This was identified as chymotrypsin.
which also binds to this affinity ligand. Studies of the factors affecting the the operation of the system showed
that there was an inherent compromise between the efficiency of product recovery and the rate of purification.
It was found that the problem of recovery were caused by the mixing of the chamber contents into a uniform
concentration. A version of the apparatus was constructed that consisted of multiple subchambers for both the
adsorption and elution steps of the process. This limited mixing and therefore allowed the formation of a concentration
gradient. This proved a success, increasing the recovery of trypsin to 75% at approximately 2 mg h-1 in an
unoptimised run. During these separations. the apparatus carried out one complete cycle in approximately
5 minutes and used in total only a small fraction of the amount of ligand of a comparable chromatography
process. It was shown to be capable of producing highly purified products in a single step from a crude feedstock.
In certain applications the rates of purification were low and belt performance deteriorated with time. These
problems may be solved by improved belt materials and optimised operating conditions.
FUTURE ACTIVITY
The new apparatus design and the new belt material with IgG and metal chelation separations have still to be
fully tested. But it is apparent that this continuous separation method is capable of efficient operation, as
shown with the trypsin purification.
Contacts
Coordinator
EC Scientific Officer
Participant
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