High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein...
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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-751 84, Uppsala, Sweden First published at the 11 th PepTalk, January 9-13, 2012 in San Diego
High-throughput screening of HIC media in PreDictor™ plates for capturing a recombinant protein from E.coli Charlotte Brink, Carina Engstrand, Eva Heldin
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-751 84, Uppsala, Sweden First published at the
11 th PepTalk, January 9-13, 2012 in San Diego
Slide 2
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.
Slide 3
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.
Slide 4
4 /High-throughput screening of HIC media in PreDictor plates
for capturing a recombinant protein from E.coli Introduction
Slide 5
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 l PreDictor HIC Screening Low Hydrophobicity, 50 l 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 L equilibration buffer, 1 min
incubation Sample loading: 200 L clarified rGFP sample (approx. 1
mg/mL), 60 min incubation Wash 1 to 3: 200 L equilibration buffer,
1 min incubation Elution 1 to 3: 200 L different elution buffers, 1
min incubation Strip: 200 l strip buffer, 1 min incubation
Evaluation: Yield and purity are determined by using GFPs specific
absorbance at 490 nm. Purity: A 490 /(A 280 -A 310 ) Purification
factor: Purity in eluate/Purity in crude sample
Slide 6
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 l PreDictor HIC Screening Low Hydrophobicity, 50 l
Slide 7
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.
Slide 8
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.
Slide 9
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.
Slide 10
10 /High-throughput screening of HIC media in PreDictor plates
for capturing a recombinant protein from E.coli Screening I
Results: Yield (cont) Fig 3b (cont). 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.
Slide 11
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
Slide 12
12 /High-throughput screening of HIC media in PreDictor plates
for capturing a recombinant protein from E.coli Screening I
Results: Purity (cont) Fig 4b (cont). 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
Slide 13
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.
Slide 14
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.
Slide 15
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.
Slide 16
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
Slide 17
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
Slide 18
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).
Slide 19
19 /High-throughput screening of HIC media in PreDictor plates
for capturing a recombinant protein from E.coli Summary
Slide 20
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.
Slide 21
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
CompanyAll 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, Bjrkgatan 30, 751 84
Uppsala, Sweden.