Designing monoclonal antibody fragment-based affinity resins with high binding capacity by thiol-directed immobilisation and optimisation of pore/ligand size ratio
Sakhnini, Laila Ismail LU
; Pedersen, Anja Kallesøe
; Ahmadian, Haleh
; Hansen, Jens Jacob
; Bülow, Leif
LU
and Dainiak, Maria
LU
(2016)
In Journal of Chromatography A
1468.
p.143-153
- Abstract
Monoclonal antibody (mAb) based affinity resins usually suffer from low binding capacity, most probably as a result of steric hindrance by the large 150 kDa size of the mAb and a random immobilisation approach. The present work investigates the influence of a variety of factors on dynamic binding capacity (DBC) such as pore/ligand size ratio, accessibility of ligand and ligand density. The effect of pore/ligand size ratio was investigated using Fab and scFv fragments on various resins with different pore sizes. The accessibility of the ligand was investigated by a site-directed immobilisation approach, where three C-terminal tags, PPKPPK, FLAG™ and Cys, were introduced into the Fab fragments for immobilisation on resins via amino-,... (More)
Monoclonal antibody (mAb) based affinity resins usually suffer from low binding capacity, most probably as a result of steric hindrance by the large 150 kDa size of the mAb and a random immobilisation approach. The present work investigates the influence of a variety of factors on dynamic binding capacity (DBC) such as pore/ligand size ratio, accessibility of ligand and ligand density. The effect of pore/ligand size ratio was investigated using Fab and scFv fragments on various resins with different pore sizes. The accessibility of the ligand was investigated by a site-directed immobilisation approach, where three C-terminal tags, PPKPPK, FLAG™ and Cys, were introduced into the Fab fragments for immobilisation on resins via amino-, carboxyl- and thiol-groups, respectively. The scFv fragments were tagged at the C-terminal only with FLAG™ to enable a straight forward purification procedure, and were immobilised to resins via amino- and carboxyl-groups. The target protein had a molecular weight (MW) of 50 kDa. A 3-fold higher dynamic binding capacity at 100% breakthrough (DBC100%) was observed for Fab wild-type (wt) on CNBr-activated Sepharose 4 FF relative to mAb on same resin at the same ligand density. However, no major difference in DBC100% was observed between Fab wt and scFv immobilised on CNBr-activated Sepharose 4 FF at the same ligand density. Thus, further increase of pore/ligand size ratio from Fab to scFv on a resin with average pore size of 300 Å, did not seem to be beneficial. Among the tested tags, only the C-terminal Cys tag proved to site-direct the ligands during immobilisation as it allowed the DBC100% to increase 1.6-fold as compared to Fab wt immobilised via amino-groups on CNBr-activated Sepharose 4 FF and Actigel ALD Superflow at the same ligand density. The influence of ligand density was investigated by selecting immobilised Fab Cys on Sulfhydryl-reactive resin. Increasing ligand density from 0.103 to 0.202 μmol/mL resulted in the same utilisation yield (82–85%), whereas a further increase in ligand density from 0.202 to 0.328 μmol/mL resulted in a 20%-unit decrease in utilisation yield and less steep breakthrough curve, suggesting steric hindrance in the pores of the resin. In addition, site-directed affinity ligands resulted in a more pronounced, sigmoid-shaped breakthrough curve, leading to more efficient use of capacity. The highest DBC100% was obtained for Fab Cys on Sulfhydryl-reactive resin and scFv on Actigel ALD Superflow; 11 mg/mL and 10 mg/mL, respectively, as compared to the DBC100% of 0.8 mg/mL for mAb on CNBr-activated Sepharose 4 FF. Pore/ligand size ratio of 3, which was achieved for Fab ligands on the studied resins, was shown to be feasible for capturing a protein in MW of 50 kDa. Totally, a 13.8-fold improvement in DBC100% was achieved with the Fab-based affinity resin coupled via the C-terminal Cys as compared to the mAb-based affinity resin.
(Less)
- author
- Sakhnini, Laila Ismail
LU
; Pedersen, Anja Kallesøe
; Ahmadian, Haleh
; Hansen, Jens Jacob
; Bülow, Leif
LU
and Dainiak, Maria
LU
- organization
- publishing date
- 2016-10-14
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Affinity resins, Dynamic binding capacity, Immobilisation yield, Ligand density, Pore/ligand size ratio, Site-directed immobilisation, Utilisation yield
- in
- Journal of Chromatography A
- volume
- 1468
- pages
- 11 pages
- publisher
- Elsevier
- external identifiers
-
- pmid:27663728
- wos:000385326500019
- scopus:84994477398
- ISSN
- 0021-9673
- DOI
- 10.1016/j.chroma.2016.09.035
- language
- English
- LU publication?
- yes
- id
- 803b0d96-8222-4ed9-bcf2-a5adfc3552f7
- date added to LUP
- 2016-12-07 10:41:15
- date last changed
- 2024-02-03 06:00:21
@article{803b0d96-8222-4ed9-bcf2-a5adfc3552f7,
abstract = {{<p>Monoclonal antibody (mAb) based affinity resins usually suffer from low binding capacity, most probably as a result of steric hindrance by the large 150 kDa size of the mAb and a random immobilisation approach. The present work investigates the influence of a variety of factors on dynamic binding capacity (DBC) such as pore/ligand size ratio, accessibility of ligand and ligand density. The effect of pore/ligand size ratio was investigated using Fab and scFv fragments on various resins with different pore sizes. The accessibility of the ligand was investigated by a site-directed immobilisation approach, where three C-terminal tags, PPKPPK, FLAG™ and Cys, were introduced into the Fab fragments for immobilisation on resins via amino-, carboxyl- and thiol-groups, respectively. The scFv fragments were tagged at the C-terminal only with FLAG™ to enable a straight forward purification procedure, and were immobilised to resins via amino- and carboxyl-groups. The target protein had a molecular weight (MW) of 50 kDa. A 3-fold higher dynamic binding capacity at 100% breakthrough (DBC<sub>100%</sub>) was observed for Fab wild-type (wt) on CNBr-activated Sepharose 4 FF relative to mAb on same resin at the same ligand density. However, no major difference in DBC<sub>100%</sub> was observed between Fab wt and scFv immobilised on CNBr-activated Sepharose 4 FF at the same ligand density. Thus, further increase of pore/ligand size ratio from Fab to scFv on a resin with average pore size of 300 Å, did not seem to be beneficial. Among the tested tags, only the C-terminal Cys tag proved to site-direct the ligands during immobilisation as it allowed the DBC<sub>100%</sub> to increase 1.6-fold as compared to Fab wt immobilised via amino-groups on CNBr-activated Sepharose 4 FF and Actigel ALD Superflow at the same ligand density. The influence of ligand density was investigated by selecting immobilised Fab Cys on Sulfhydryl-reactive resin. Increasing ligand density from 0.103 to 0.202 μmol/mL resulted in the same utilisation yield (82–85%), whereas a further increase in ligand density from 0.202 to 0.328 μmol/mL resulted in a 20%-unit decrease in utilisation yield and less steep breakthrough curve, suggesting steric hindrance in the pores of the resin. In addition, site-directed affinity ligands resulted in a more pronounced, sigmoid-shaped breakthrough curve, leading to more efficient use of capacity. The highest DBC<sub>100%</sub> was obtained for Fab Cys on Sulfhydryl-reactive resin and scFv on Actigel ALD Superflow; 11 mg/mL and 10 mg/mL, respectively, as compared to the DBC<sub>100%</sub> of 0.8 mg/mL for mAb on CNBr-activated Sepharose 4 FF. Pore/ligand size ratio of 3, which was achieved for Fab ligands on the studied resins, was shown to be feasible for capturing a protein in MW of 50 kDa. Totally, a 13.8-fold improvement in DBC<sub>100%</sub> was achieved with the Fab-based affinity resin coupled via the C-terminal Cys as compared to the mAb-based affinity resin.</p>}},
author = {{Sakhnini, Laila Ismail and Pedersen, Anja Kallesøe and Ahmadian, Haleh and Hansen, Jens Jacob and Bülow, Leif and Dainiak, Maria}},
issn = {{0021-9673}},
keywords = {{Affinity resins; Dynamic binding capacity; Immobilisation yield; Ligand density; Pore/ligand size ratio; Site-directed immobilisation; Utilisation yield}},
language = {{eng}},
month = {{10}},
pages = {{143--153}},
publisher = {{Elsevier}},
series = {{Journal of Chromatography A}},
title = {{Designing monoclonal antibody fragment-based affinity resins with high binding capacity by thiol-directed immobilisation and optimisation of pore/ligand size ratio}},
url = {{http://dx.doi.org/10.1016/j.chroma.2016.09.035}},
doi = {{10.1016/j.chroma.2016.09.035}},
volume = {{1468}},
year = {{2016}},
}