development of capture and intermediate chromatography ...purification introduction • this poster...

Development of capture and intermediate chromatography

steps for insulin purification

J. Shanagar*1, D. Krishnan1, E.Heldin1, S. Grönlund1, E. Hallgren1, K. Eriksson1, H. Tunes2, M. Xavier2, L. Vilela2 1GE Healthcare Bio-Sciences AB, Uppsala, Sweden and 2BIOMM S.A. Belo Horizonte, Brazil *E-mail: [email protected]

First presented at the 241st ACS Spring 2011 National Meeting & Exposition, Anaheim, CA, March 27–31, 2011.

2 /Development of capture and intermediate chromatography steps for insulin

purification

Summary •  A capture step for pro-insulin expressed in E. coli and an

intermediate step for the purification of insulin following the capture step were developed.

•  Resin screening and chromatography condition screening using an HTPD approach in a 96-well filter-plate format were performed.

•  The results indicated that the media of choice for the capture step was Capto™ MMC, a multi-modal cation-exchange resin.

•  Elution conditions were further optimized in packed columns using the Design of Experiments (DoE) functionality in an ÄKTA™ avant 25 chromatography system.


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Summary •  The optimal chromatographic conditions were identified and

followed by a robustness study.

•  The capture step was then scaled to pilot scale.

•  After enzymatic cleavage of the purified pro-insulin from the capture step the formed insulin was subjected to cation exchange chromatography.

•  A similar approach for development of this step as for the capture step was followed.

•  The results indicated that the best media for the intermediate step was Capto ™ SP ImpRes.

•  Robustness and scale up to pilot scale were performed.


purification

Introduction •  This poster illustrates a case study for the development of the capture purification

step for a recombinant pro-insulin expressed in E. coli, and an intermediate step for the purification of insulin following the capture step.

•  Both chromatographic resin and operating conditions were screened and optimized.

•  Pro-insulin and cleaved insulin structures are shown in Figure 1. The molecular weight of pro-insulin and insulin are ~11,000 and ~ 5800, respectively and the pI’s are 5.6.


purification

Introduction Figure 2 shows the work flow for the process development and the scale up to a small Pilot scale.


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Screening method with PreDictor™ plates

Resin screening, as well as condition screening for binding and elution were done with a High Throughput Process Development (HTPD) approach, using the PreDictor plate format (Figure 3).


purification

Results using PreDictor™ plates Resin screening showed that Capto™ MMC was best for the capture step especially since the sample could be loaded without dilution or an UF/DF step (i.e. loading in 8 M urea at ~ 15mS/cm). The structure of the MMC ligand is shown in Figure 4. The screening also showed that Capto SP ImpRes and SP Sepharose™ Fast Flow were best resins for the intermediate step. Binding and elution conditions for both steps were also screened in the PreDictor plate format. Results are shown in Figure 5 (intermediate step not shown). Optimal loading conditions for capture step should be around pH 5.2 and elution above pH 6.2 and with a NaCl concentration above 400 mM.


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Analysis

Analysis was done using a Mono Q™ column for the capture step and a C4-100 Kromasil® RPC column for the intermediate step.


purification

10 /Development of capture and intermediate chromatography steps for

insulin purification



Conclusions • The conditions were first identified in the PreDictor™ format

and successfully transferred to column format.

• The best media for capture & intermediate steps were found to be Capto™ MMC & Capto SP ImpRes, respectively.

• Optimization and robustness studies was done with an ÄKTA™ avant 25 system.

• Both the capture- and Intermediate purification steps were successfully scaled 400 fold.

• The purity of the pro-Insulin and insulin was 82% and 92%, respectively with recovery of > 95% in both steps.



Acknowledgments

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