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FAIR-CT95-1039
Processing Technology for Recovery of Recombinant Antibody Produced in Crop Plants |
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Proposal No: | FAIR-CT95-1039 |
| Date Prepared: | April 1998, September 1999 | |
| Source: | Second Annual Progress Report First Annual Progress Report |
Objectives
The key objectives for Year 2 of the project were
to deliver antibody fragments for FSH and GOx synthesised in microbial
hosts on a laboratory scale and complementary peptide ligands designed for
affinity chromatography. This would allow the investigation, on a
laboratory scale, of the possibility of exploiting affinity chromatography
for purification of antibody fragments from plant tissues and thereby lay
the foundation for pilot scale development. The ultimate objective in Year
3 is to separate antibody fragments from plant tissue systems of
production either in plant suspension culture or growing plants.
Description of Work
Ligands
Libraries of candidate ligands for
FSH and GOx have been developed by 'Replacement Net Pepscan' from lead
sequences derived initially from Pepscan arrays or from phage displayed
random peptide libraries. The affinity binding characteristics of the
ligands have been evaluated for the antibodies by conventional ELISA and
also by the BIAcore system with the peptide ligands presented on the solid
phase.
Selected sequences have been further improved by rational design in order to improve mAb binding and also solid phase characteristics. From this point selected peptides have also been synthesised on milligram scale for use in development of affinity chromatography systems. The benchmark for the laboratory scale evaluations was Sepharose.
Recombinant Antibody Systems
Direct cloning
from hybridoma cell lines as well as combinatorial phage displayed
libraries from immunised mice has been established to select for scFv's
directed against the antigen of choice, FSH and GOx. Genes for scFv's have
been subcloned for stable expression as soluble protein, first in E.coli
and subsequently for production in practicable amounts in yeast (Pichia
pastoris)
Following confirmation of specificity and ligand binding the Fv genes have been introduced into appropriate vectors for transformation of plant cells (entering Task 5, Year3).
State of Progress
The crucial issue at the
beginning of Year 2 was the question of the format of the peptide ligands
for the appropriate solid phase reaction with the monoclonal antibodies.
This was focal to the development of the affinity process which is the key
objective of the project. This issue has been completely resolved for FSH
for which working chromatography systems are in use on a laboratory scale.
Peptides and scFv's are now also available for GOx, with evaluation of the
affinity process on a laboratory scale in progress. We have established
competencies in every aspect of technology which we defined as necessary
for the successful progress of the project and in consequence have met key
milestones and deliverables for advancing the concepts.
In brief, against the projected deliverables and milestones the status is as follows.
| Code letter | Timing (Task linkage) |
Description of technical deliverables and milestones | Status |
| A* | 6 months (enters T2 from T1) |
Selected linear peptides and immunoassays for FSH and GOx based on polyclonal serum antibodies | Complete |
| B | 9 months (enters T3 from T1) |
Phages displaying panel of Fvs binding to FSH and GOx | Complete |
| C | 12 months (enters T2 and T3 from T1) |
Monoclonal antibodies binding to FSH and GOx peptides and their hybridomas | Complete |
| D* | 15 months (enters T2 from T3) |
Antibody fragments synthesised in microbial hosts (laboratory scale) | Complete |
| E | 18 months (enters T5 from T3) |
Fv genes in appropriate vectors for transformation of plant cells | Complete |
| F* | 24 months (enters T4 from T2) |
Laboratory scale affinity chormatography qualified by MAbs microbially derived Fvs. Processes for pilot-scale development | FSH complete GOx ongoing |
| G | 24 months (enters T4 from T5) |
Microbial culture supernatants for initial pilot-scale development of Fv affinity processing | FSH complete GOx ongoing |
* Denotes a major milestone
Achievements
A prospective affinity process
which lays the foundation for the purification of recombinant antibodies
has been established. All the key technologies are in place for laboratory
scale-up to a pilot scale activity in Year 3. Peptide ligands are defined
and coupled to the conventional solid phase matrix Sepharose. Other
matrices are under investigation. Recombinant anti-FSH and anti-GOx
antibodies are being produced in Pichia pastoris in laboratory scale
fermentors so that pilot scale development of the affinity processes can
take place. The scFv genes for FSH have been expressed in plants with work
on GOx in progress. Immunoassays are in place, but need to be optimised,
for rigorous QC of the systems.
Future Actions
We have established a strong
learning curve with FSH antibodies and peptide ligands; this being closely
followed up with GOx. The immediate objective with both systems is to
consolidate our experience on solid phase ligand coupling, loading
efficiency, stability and reusability in order to enter the scale-up phase
of process development required in Task 4. Yeast production of scFv's in
fermentors is in hand to provide necessary quantities of antibody
fragments for plant cell spiking studies for process development. The
final phase of the technology will be the production of the recombinant
antibodies in plant expression systems as a key deliverable for assessment
of the pilot scale affinity process. Thereafter with all procedures of
purification and QC in place there will be a final evaluation of the
technology.
Objectives
The key objective is to provide generic processing
technologies for affinity-based separation of antibody fragments produced in
plants, by exploiting small stable peptides which resemble the natural epitope
recognised by the antibody. The peptides will be selected on the basis that they
are inexpensive to produce in bulk, and designed with the requirements of being
linked with large scale supports to allow quick, reliable recovery and also
multiple release of the affinity medium.
Description of work
The process development will be modelled
using antibody fragments developed against two antigens eg. a peptide hormone
(FSH) and an enzyme glucose oxidase (GOx). The following activities will be
undertaken. Peptide antigen analogues or epitopes will be defined as binding
ligands for the antibodies and Ig fragments by technologies such as phage
displayed random peptide libraries or Pepscan arrays of linear epitopes. Genes
of appropriately designed antibody fragments which bind these ligands will be
transfected into yeast, tobacco and potato to provide material on a practical
scale for process development. Specific affinity purification media, based on
appropriately designed peptide antigen analogues, will be developed with a view
to capture and release of yeast and plant produced antibody fragments in a
stable and multiple reusable system. Process control and product QC techniques
will be developed to monitor and validate the process. A working pilot process
will be evolved for which the performance characteristics will be assessed in
terms of product yield, reusability/stability, cost efficiency and ease of
further scale up and downstream processing.
Progress
To date the project is very much on target and the
deliverables sustained. Selected linear peptides binding in immunoassays for FSH
and GOx have been defined for polyclonal serum antibodies and monoclonal
antibodies are in preparation from hybridomas. Direct cloning of antibody
fragments from phages displaying scFvs is being undertaken. These are the key
technical deliverables meeting milestones in the first 12 months of the project.
In addition, fermentation equipment for both yeast and plant cells has been
purchased and setup. Fermentation runs using model antibodies have been
successfully carried out in Pichia and tobacco cells.
Achievements
Monoclonal antibodies against the model antigens
GOx and FSH have been produced and evaluated for peptide ligands by both Pepscan
assay and Phage displayed random peptide libraries. From linear epitope
sequences for GOx and FSH antibodies appropriate peptides have been synthesised
and checked for reactions m Immunoassays appropriate to the prospective affinity
processes to be designed for purification technology. An issue has arisen as to
whether peptides should be linear or cyclised for most appropriate binding
reaction affinity. Combinatorial phage rabbit and chicken antibody libraries are
being established from immunocompetent mice to select phages producing antibody
fragments that react with GOx/FSH peptide ligands selected above. Studies are
also ongoing with rabbits and chickens to examine their relevance as sources of
antibody genes. Vectors for cloning and expression of scFvs in the yeast Pichia
pastoris and plants have been prepared for future use in optimising
heterologous expression of suitable scFvs. Fermenters have been commissioned and
production of mouse scFv antibody fragments successfully achieved in quantities
that will facilitate the development of affinity processing media.
Future activities
A crucial issue is the question of the format
of peptide ligands ie linear or cyclised to achieve efficient binding. This will
be evaluated immediately as part of Task 2 which focuses on the development of
the affinity process. Alongside this activity will be the formatting of scFv
genes for expression in the methylotrophic yeast Pichia and also in
plants. Production of useful quantities of scFv in Pichia fermentation
will go hand in hand with optimisation and scale up of affinity process
technology in Year 2.
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Updated
by CPL Press:
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