Highthroughput screening of HIC media in Pre Dictor

High-throughput screening of HIC media in Pre. Dictor™ 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-751 84, Uppsala, Sweden First published at the 11 th Pep. Talk, January 9 -13, 2012 in San Diego

Introduction • Hydrophobic interaction chromatography (HIC) is a powerful purification technique where the type and density of the ligand, p. H 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. 2 /High-throughput screening of HIC media in Pre. Dictor™ plates for capturing a recombinant protein from E. coli

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

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

Pre. Dictor™ plate experimental procedure Pre. Dictor HIC Screening High Hydrophobicity, 50 μl Pre. Dictor 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 r. GFP sample (approx. 1 mg/m. L), 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: 490 nm. Yield and purity are determined by using GFP’s specific absorbance at Purity: A 490/(A 280 -A 310) Purification factor: Purity in eluate/Purity in crude sample 5 /High-throughput screening of HIC media in Pre. Dictor™ plates for capturing a recombinant protein from E. coli

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

Solubility test • Prior to screening, the solubility of r. GFP was measured by light scattering at 350 nm in the presence of ammonium sulphate, sodium sulphate and sodium chloride at six concentrations and p. H 7 in 96 -well UV-readable collection plates. Fig 1. Solubility of r. GFP in clarified cell culture supernatant at p. H 7 with three different salt types. 7 /High-throughput screening of HIC media in Pre. Dictor™ 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. 8 /High-throughput screening of HIC media in Pre. Dictor™ plates for capturing a recombinant protein from E. coli

Screening I Results: Yield Fig 3 a (continues on next slide). r. GFP 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 r. GFP is too strong under the conditions tested. 9 /High-throughput screening of HIC media in Pre. Dictor™ plates for capturing a recombinant protein from E. coli

Screening I Results: Yield (con’t) Fig 3 b (con’t). r. GFP 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 r. GFP is too strong under the conditions tested. 10 /High-throughput screening of HIC media in Pre. Dictor™ plates for capturing a recombinant protein from E. coli

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

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

Screening I Results: Interpretation of purity data • Difference in purification factor from plate experiments indicates selectivity between target protein and impurities. Fig 5. Comparison of plate and column data. A) purification factor as a function of salt concentration in elution. B) corresponding chromatogram 13 /High-throughput screening of HIC media in Pre. Dictor™ 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 m. L medium). The green curve is the detection of r. GFP at its unique wavelength of absorbance, 490 nm. The peak in the beginning is r. GFP-related impurities and the sharp peak at the end of the chromatogram is strongly-bound components eluted in 30% isopropanol. 14 /High-throughput screening of HIC media in Pre. Dictor™ 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 (Na. Cl) was tested; at concentrations of 2 M, 2. 5 M and 3 M Na. Cl. 15 /High-throughput screening of HIC media in Pre. Dictor™ plates for capturing a recombinant protein from E. coli

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

Screening II Results: Purity • Lower concentrations of Na. Cl in binding buffer reduce the risk of an impurity co-eluting with r. GFP Fig 9. r. GFP purity data from elution fractions. 17 /High-throughput screening of HIC media in Pre. Dictor™ 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 m. L medium columns). 18 /High-throughput screening of HIC media in Pre. Dictor™ plates for capturing a recombinant protein from E. coli

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

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

Acknowledgments GE, imagination at work, and GE monogram are trademarks of General Electric Company. Capto, Pre. Dictor, 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 2011. 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, 751 84 Uppsala, Sweden. 21 /High-throughput screening of HIC media in Pre. Dictor™ plates for capturing a recombinant protein from E. coli