Download Evaluation of novel affinity bio-beads for use in the production of

Evaluation of novel affinity bio-beads for use
in the production of plasma derived albumin.
Sergio Pagliazzi, Karl McCann, Jose Martinez, Tracy Thompson, Sara Ladd,
Joseph Bertolini.
CSL Behring, Camp Rd. Broadmeadows, Victoria
PolyBatics, Ltd. BioCommerce Centre, Palmerston North, New Zealand
Introduction
Results
The purification of albumin at CSL Behring from human plasma
involves a combination of chromatographic steps with a final gel
filtration polishing step used to purify material derived from ion
exchange chromatography. This requires multiple cycles to process a
batch and offers an opportunity to improve process efficiency. The aim
of the project was to find an alternative to this process step.
Polybind-Z
Polybind-Z was able to remove up to 99% of IgG present in the
albumin feedstock up to a loading a resin to feedstock ratio of 1:400,
while for IgA and IgM was significantly lower.
An affinity chromatography process involving the use of bio-beads,
Polybind-Z and Polybind-L, manufactured by Polybatics, NZ was
evaluated to remove the main residual proteins IgG, IgA and IgM.
Bio-beads are produced in bacteria and consist of specific ligands
incorporated onto a polyester backbone. The specific resins evaluated
were Polybind-Z with a specific affinity for IgG, and Polybind-L,
specific for the kappa light chain and able to interact with IgG, IgA and
IgM.
Structure of biobeads
produced by Polybatics,
NZ, showing ligands on
the surface of the
polyester scaffold
Polybind-L
Polybind-L had a broader affinity for immunoglobulins. It bound 70% of
IgG and IgA at a low resin to volume ratio (1:10) with increasing drop
through evident with further loading. Thus at a 1:400 resin to volume
ratio, only 30% of IgG and IgA bound to resin, indicating a progressive
saturation of binding sites. This resin had greater specificity and
capacity for IgM with up to 70% removal at a resin to volume ratio of
1:200.
Method
Resins were tested to investigate their IgG, IgA and IgM binding
capacity and specificity.
Concentrated and pH adjusted albumin process intermediate was
mixed with Polybind-Z or Polybind-L beads at various resin to
feedstock ratios. One gram of resin was used in all studies.
After thoroughly mixing the solution of resin and feedstock, the
mixture was separated by centrifugation at 17,000x g. The
supernatant was tested for unbound IgG, IgA and IgM.
Application in bioprocessing
Further process development would need to focus on establishing an appropriate means of removing the biobeads from the treated
intermediate. As centrifugation is not practical in any large scale application, the use of depth filtration coupled with use of a filter aid has
provided promising results in initial studies.
Conclusions
1. The capability of two novel affinity resins (Polybind-Z and Polybind-L) produced by bacteria and incorporated with specific ligands for IgG,
IgA and IgM was examined for the capability of removing these residual proteins an albumin intermediate containing these residual proteins.
2. Polybind-Z exhibited a high binding capacity and specificity for IgG, but low specificity for IgA and IgM when used to purify an albumin
intermediate. Polybind-L had specificity and capacity for IgM but had lower capacity for IgA and IgG.
3. Biobeads with specific ligands have the capability of removing significant amounts of trace immunoglobulin contaminants from a
predominantly albumin matrix and offer the potential to be used in an alternative low cost method for polishing an in-process albumin
intermediate.