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Optimizing selectivity of anion hydrophobic multimodal chromatography for purification of a single-chain variable fragment

Sakhnini, Laila I. LU orcid ; Pedersen, Anja K. ; León, Ileana Rodríguez ; Greisen, Per J. ; Hansen, Jens Jacob ; Vester-Christensen, Malene B. ; Bülow, Leif LU and Dainiak, Maria B. LU (2019) In Engineering in Life Sciences 19(7). p.490-501
Abstract

Single-chain variable fragments (scFv) are widely used in several fields. However, they can be challenging to purify unless using expensive Protein L-based affinity adsorbents or affinity tags. In this work, a purification process for a scFv using mixed-mode (MM) chromatography was developed by design of experiments (DoE) and proteomics for host cell protein (HCP) quantification. Capture of scFv from human embryonic kidney 293 (HEK293) cell feedstocks was performed by hydrophobic charge induction chromatography (MEP HyperCel™), whereafter polishing was performed by anion hydrophobic MM chromatography (Capto Adhere™). The DoE designs of the polishing step included both binding and flow-through modes, the latter being the standard mode... (More)

Single-chain variable fragments (scFv) are widely used in several fields. However, they can be challenging to purify unless using expensive Protein L-based affinity adsorbents or affinity tags. In this work, a purification process for a scFv using mixed-mode (MM) chromatography was developed by design of experiments (DoE) and proteomics for host cell protein (HCP) quantification. Capture of scFv from human embryonic kidney 293 (HEK293) cell feedstocks was performed by hydrophobic charge induction chromatography (MEP HyperCel™), whereafter polishing was performed by anion hydrophobic MM chromatography (Capto Adhere™). The DoE designs of the polishing step included both binding and flow-through modes, the latter being the standard mode for HCP removal. Chromatography with Capto Adhere™ in binding-mode with elution by linear salt gradient at pH 7.5 resulted in optimal yield, purity and HCP reduction factor of 98.9 > 98.5%, and 14, respectively. Totally, 258 different HCPs were removed, corresponding to 84% of identified HCPs. The optimized conditions enabled binding of the scFv to Capto Adhere™ below its theoretical pI, while the majority of HCPs were in the flow-through. Surface property maps indicated the presence of hydrophobic patches in close proximity to negatively charged patches that could potentially play a role in this unique selectivity.

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author
; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
mixed-mode chromatography, process development, proteomics, single-chain variable fragment, spatial aggregation propensity
in
Engineering in Life Sciences
volume
19
issue
7
pages
490 - 501
publisher
Wiley-Blackwell
external identifiers
  • scopus:85065655964
  • pmid:32625026
ISSN
1618-0240
DOI
10.1002/elsc.201800207
language
English
LU publication?
yes
id
30ef466a-995a-4b07-94da-b7605a00bfd7
date added to LUP
2019-06-17 14:16:44
date last changed
2024-10-02 05:00:39
@article{30ef466a-995a-4b07-94da-b7605a00bfd7,
  abstract     = {{<p>Single-chain variable fragments (scFv) are widely used in several fields. However, they can be challenging to purify unless using expensive Protein L-based affinity adsorbents or affinity tags. In this work, a purification process for a scFv using mixed-mode (MM) chromatography was developed by design of experiments (DoE) and proteomics for host cell protein (HCP) quantification. Capture of scFv from human embryonic kidney 293 (HEK293) cell feedstocks was performed by hydrophobic charge induction chromatography (MEP HyperCel™), whereafter polishing was performed by anion hydrophobic MM chromatography (Capto Adhere™). The DoE designs of the polishing step included both binding and flow-through modes, the latter being the standard mode for HCP removal. Chromatography with Capto Adhere™ in binding-mode with elution by linear salt gradient at pH 7.5 resulted in optimal yield, purity and HCP reduction factor of 98.9 &gt; 98.5%, and 14, respectively. Totally, 258 different HCPs were removed, corresponding to 84% of identified HCPs. The optimized conditions enabled binding of the scFv to Capto Adhere™ below its theoretical pI, while the majority of HCPs were in the flow-through. Surface property maps indicated the presence of hydrophobic patches in close proximity to negatively charged patches that could potentially play a role in this unique selectivity.</p>}},
  author       = {{Sakhnini, Laila I. and Pedersen, Anja K. and León, Ileana Rodríguez and Greisen, Per J. and Hansen, Jens Jacob and Vester-Christensen, Malene B. and Bülow, Leif and Dainiak, Maria B.}},
  issn         = {{1618-0240}},
  keywords     = {{mixed-mode chromatography; process development; proteomics; single-chain variable fragment; spatial aggregation propensity}},
  language     = {{eng}},
  number       = {{7}},
  pages        = {{490--501}},
  publisher    = {{Wiley-Blackwell}},
  series       = {{Engineering in Life Sciences}},
  title        = {{Optimizing selectivity of anion hydrophobic multimodal chromatography for purification of a single-chain variable fragment}},
  url          = {{http://dx.doi.org/10.1002/elsc.201800207}},
  doi          = {{10.1002/elsc.201800207}},
  volume       = {{19}},
  year         = {{2019}},
}