High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Charlotte Brink, Carina Engstrand, Eva Heldin and Susanne Nyholm Westin GE Healthcare Bio-Sciences AB, SE , Uppsala, Sweden First published at the 11 th PepTalk, January 9-13, 2012 in San Diego

2 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Introduction Hydrophobic interaction chromatography (HIC) is a powerful purification technique where the type and density of the ligand, pH and salt of binding conditions, temperature and the nature of the target protein are highly significant parameters in finding and fine tuning selectivity. A parallel screening of HIC media and conditions in 96-well plates facilitates the selection of the most promising HIC media and shows how salt type and salt concentration conditions could be tailored to successfully capture the target compound.

3 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Introduction In this study the model compound is Green Fluorescent Protein (rGFP) expressed in E. coli. The plate results are compared with those obtained using traditional packed-bed chromatography columns.

4 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Introduction

5 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli PreDictor™ plate experimental procedure PreDictor HIC Screening High Hydrophobicity, 50 μl PreDictor HIC Screening Low Hydrophobicity, 50 μl Batch uptake experiments were executed according to the instructions and with the help of Assist software. The experimental steps were as follows: Equilibration 1 to 3: 200 μL equilibration buffer, 1 min incubation Sample loading: 200 μL clarified rGFP sample (approx. 1 mg/mL), 60 min incubation Wash 1 to 3: 200 μL equilibration buffer, 1 min incubation Elution 1 to 3: 200 μL different elution buffers, 1 min incubation Strip: 200 μl strip buffer, 1 min incubation Evaluation: Yield and purity are determined by using GFP’s specific absorbance at 490 nm. Purity: A 490 /(A 280 -A 310 ) Purification factor: Purity in eluate/Purity in crude sample

6 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli PreDictor™ plate experimental procedure PreDictor HIC Screening High Hydrophobicity, 50 μl PreDictor HIC Screening Low Hydrophobicity, 50 μl

7 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Solubility test Prior to screening, the solubility of rGFP was measured by light scattering at 350 nm in the presence of ammonium sulphate, sodium sulphate and sodium chloride at six concentrations and pH 7 in 96-well UV-readable collection plates. Fig 1. Solubility of rGFP in clarified cell culture supernatant at pH 7 with three different salt types.

8 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Screening I Initial media and condition screening in a parallel format will enable a rapid way of investigating many parameters and efficiently screen for the required selectivity. An elution study with varied elution conditions may thus reveal conditions where more target protein is eluted then HCPs. Fig 2. Plate design for screening media and salt type viewed for the low hydrophobicity screening plate. Same distribution of factors were used for the high hydrophobicity screening plate.

9 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Screening I Results: Yield Fig 3a (continues on next slide). rGFP yield data (from elution fractions). Low yield is seen for: Capto™ Butyl, Phenyl Sepharose™ 6 Fast Flow (high sub), and Capto Phenyl (high sub) indicating that the hydrophobic interaction of rGFP is too strong under the conditions tested.

10 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Screening I Results: Yield (con’t) Fig 3b (con’t). rGFP yield data (from elution fractions). Low yield is seen for: Capto™ Butyl, Phenyl Sepharose™ 6 Fast Flow (high sub), and Capto Phenyl (high sub) indicating that the hydrophobic interaction of rGFP is too strong under the conditions tested.

11 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Screening I Results: Purity Fig 4a. rGFP purity data from elution fractions from Butyl-S Sepharose ™ 6 Fast Flow. First screening revealed 4 interesting HIC media (see Fig 4b). The chromatographic results correlate well with the purity revealed in the plate experiments

12 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Screening I Results: Purity (con’t) Fig 4b (con’t). rGFP purity data from elution fractions. First screening revealed 4 interesting HIC media. The chromatographic results correlate well with the purity revealed in the plate experiments

13 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Screening I Results: Interpretation of purity data Fig 5. Comparison of plate and column data. A) purification factor as a function of salt concentration in elution. B) corresponding chromatogram Difference in purification factor from plate experiments indicates selectivity between target protein and impurities.

14 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Screening I Results: Comparison with chromatography columns Fig 6. Chromatograms of Tricorn™ 5/50 runs (1 mL medium). The green curve is the detection of rGFP at its unique wavelength of absorbance, 490 nm. The peak in the beginning is rGFP-related impurities and the sharp peak at the end of the chromatogram is strongly-bound components eluted in 30% isopropanol.

15 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Screening II The first screening revealed a possibility to remove impurities into the flowthrough. In the second screening the effect on selectivity fraction by using lower salt concentration for binding was investigated. For the sake of simplicity, only one salt type (NaCl) was tested; at concentrations of 2M, 2.5M and 3M NaCl.

16 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Screening II Results: Yield Fig 8. rGFP yield data from elution fractions. Lower concentrations of NaCl in binding buffer reduce the risk of an impurity co-eluting with rGFP

17 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Screening II Results: Purity Fig 9. rGFP purity data from elution fractions. Lower concentrations of NaCl in binding buffer reduce the risk of an impurity co-eluting with rGFP

18 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Chromatograms from two of the most promising HIC media Fig 10. Chromatograms of Tricorn 5/50 runs (1 mL medium columns).

19 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Summary

20 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Total time and sample amounts consumed Fig 11. Estimate of time and sample consumption for PreDictor™ HIC screening plates compared with column chromatography.

21 /High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Acknowledgments GE, imagination at work, and GE monogram are trademarks of General Electric Company. Capto, PreDictor, Sepharose and Tricorn are trademarks of GE Healthcare companies. All third party trademarks are property of their respective owners. © 2012 General Electric Company―All rights reserved. First published All goods and services are sold subjects to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare Bio-Sciences AB, Björkgatan 30, Uppsala, Sweden.