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Antibody-Fragments: Protein Engineering, Process Development, and their Application as Affinity Ligands

Sakhnini, Laila LU orcid (2019)
Abstract
Monoclonal antibody (mAb) fragments have become the formats of choice for both biopharmaceuticals and ligands in analytical and preparative applications. Their smaller size enables additional advantages over full-length mAbs, such as improved tissue penetration, faster blood clearance for diagnostic applications, improved capacity, and enablement of multivalent formatting. However, limitations in their biophysics and downstream processing can become a challenge in their development. In this thesis, three different aspects of these limitations have been investigated.

First, a strategy to improve the developability potential of a model antigen binding fragment (Fab) was investigated. 393 Fab variants with single and double/triple... (More)
Monoclonal antibody (mAb) fragments have become the formats of choice for both biopharmaceuticals and ligands in analytical and preparative applications. Their smaller size enables additional advantages over full-length mAbs, such as improved tissue penetration, faster blood clearance for diagnostic applications, improved capacity, and enablement of multivalent formatting. However, limitations in their biophysics and downstream processing can become a challenge in their development. In this thesis, three different aspects of these limitations have been investigated.

First, a strategy to improve the developability potential of a model antigen binding fragment (Fab) was investigated. 393 Fab variants with single and double/triple mutations in the frameworks and complementarity determining regions were screened by in silico computational models. Out of this library, 26 variants were selected for in vitro developability assessment. A majority of the variants exhibited same or improved thermodynamic stability relative to wild-type. In addition, they were improved with regards to non-specificity. Variants containing [28D]-FR1-H, [31D]-CDR1-H and [53D]-CDR2-L showed to be improved in regard to aggregation propensity. Remarkably, the affinity to the target protein was fully retained in all variants.

Second, a downstream process was developed for a single-chain variable fragment (scFv) with kappa light chain II, which cannot be purified by conventional methods. Design of experiments was employed together with a proteomics approach for identification and relative quantitation of host cell protein (HCP). Capture and polishing were performed by hydrophobiccharge induction multimodal chromatography (MMC) and anion hydrophobic MMC, respectively. The polish step was developed and optimised by screening and optimisation DoE. Operational parameters were successfully screened, and optimal purity, yield, and HCP reduction factor were estimated to >98 %, >98 %, and 14, respectively.

Third, a systematic study for the development of high-capacity immunoaffinity adsorbents was conducted. MAb, Fab, scFv and multimeric scFv fusions were investigated as affinity ligands. The accessibility of the binding sites had substantial effect on the binding capacity. It turned out to be largely impacted by the ligand format, ligand density, and orientation of ligands. Altogether, highest binding capacity was achieved for adsorbents with site-directed monomeric and multimeric scFv ligands; up to 20 mg/mL for a target protein of 50 kDa. (Less)
Abstract (Swedish)
Monoclonal antibody (mAb) fragments have become the formats of choice for both biopharmaceuticals and ligands in analytical and preparative applications. Their smaller size enables additional advantages over full-length mAbs, such as improved tissue penetration, faster blood clearance for diagnostic applications, improved capacity, and enablement of multivalent formatting. However, limitations in their biophysics and downstream processing can become a challenge in their development. In this thesis, three different aspects of these limitations have been investigated.

First, a strategy to improve the developability potential of a model antigen binding fragment (Fab) was investigated. 393 Fab variants with single and double/triple... (More)
Monoclonal antibody (mAb) fragments have become the formats of choice for both biopharmaceuticals and ligands in analytical and preparative applications. Their smaller size enables additional advantages over full-length mAbs, such as improved tissue penetration, faster blood clearance for diagnostic applications, improved capacity, and enablement of multivalent formatting. However, limitations in their biophysics and downstream processing can become a challenge in their development. In this thesis, three different aspects of these limitations have been investigated.

First, a strategy to improve the developability potential of a model antigen binding fragment (Fab) was investigated. 393 Fab variants with single and double/triple mutations in the frameworks and complementarity determining regions were screened by in silico computational models. Out of this library, 26 variants were selected for in vitro developability assessment. A majority of the variants exhibited same or improved thermodynamic stability relative to wild-type. In addition, they were improved with regards to non-specificity. Variants containing [28D]-FR1-H, [31D]-CDR1-H and [53D]-CDR2-L showed to be improved in regard to aggregation propensity. Remarkably, the affinity to the target protein was fully retained in all variants.

Second, a downstream process was developed for a single-chain variable fragment (scFv) with kappa light chain II, which cannot be purified by conventional methods. Design of experiments was employed together with a proteomics approach for identification and relative quantitation of host cell protein (HCP). Capture and polishing were performed by hydrophobiccharge induction multimodal chromatography (MMC) and anion hydrophobic MMC, respectively. The polish step was developed and optimised by screening and optimisation DoE. Operational parameters were successfully screened, and optimal purity, yield, and HCP reduction factor were estimated to >98 %, >98 %, and 14, respectively.

Third, a systematic study for the development of high-capacity immunoaffinity adsorbents was conducted. MAb, Fab, scFv and multimeric scFv fusions were investigated as affinity ligands. The accessibility of the binding sites had substantial effect on the binding capacity. It turned out to be largely impacted by the ligand format, ligand density, and orientation of ligands. Altogether, highest binding capacity was achieved for adsorbents with site-directed monomeric and multimeric scFv ligands; up to 20 mg/mL for a target protein of 50 kDa. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Associate Professor van Alstine, James, Royal Institute of Technology, Stockholm.
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Monoclonal Antibodies, Aggregation, Biophysical Characterisation, Process Development, Affinity Adsorbents, Monoclonal Antibodies, Aggregation, Biophysical Characterisation, Process Development, Affinity Adsorbents
pages
80 pages
publisher
Lund University, Faculty of Engineering
defense location
Lecture hall KC:B, Kemicentrum, Naturvetarvägen 14, Faculty of Engineering LTH, Lund
defense date
2019-11-18 13:00:00
ISBN
978-91-7422-701-7
978-91-7422-700-0
language
English
LU publication?
yes
id
3156874d-3d8d-41ad-bd7a-6be6ffb8e037
date added to LUP
2019-10-23 12:02:40
date last changed
2020-12-03 12:34:16
@phdthesis{3156874d-3d8d-41ad-bd7a-6be6ffb8e037,
  abstract     = {{Monoclonal antibody (mAb) fragments have become the formats of choice for both biopharmaceuticals and ligands in analytical and preparative applications. Their smaller size enables additional advantages over full-length mAbs, such as improved tissue penetration, faster blood clearance for diagnostic applications, improved capacity, and enablement of multivalent formatting. However, limitations in their biophysics and downstream processing can become a challenge in their development. In this thesis, three different aspects of these limitations have been investigated. <br>
<br>
First, a strategy to improve the developability potential of a model antigen binding fragment (Fab) was investigated. 393 Fab variants with single and double/triple mutations in the frameworks and complementarity determining regions were screened by in silico computational models. Out of this library, 26 variants were selected for in vitro developability assessment. A majority of the variants exhibited same or improved thermodynamic stability relative to wild-type. In addition, they were improved with regards to non-specificity. Variants containing [28D]-FR1-H, [31D]-CDR1-H and [53D]-CDR2-L showed to be improved in regard to aggregation propensity. Remarkably, the affinity to the target protein was fully retained in all variants. <br>
<br>
Second, a downstream process was developed for a single-chain variable fragment (scFv) with kappa light chain II, which cannot be purified by conventional methods. Design of experiments was employed together with a proteomics approach for identification and relative quantitation of host cell protein (HCP). Capture and polishing were performed by hydrophobiccharge induction multimodal chromatography (MMC) and anion hydrophobic MMC, respectively. The polish step was developed and optimised by screening and optimisation DoE. Operational parameters were successfully screened, and optimal purity, yield, and HCP reduction factor were estimated to &gt;98 %, &gt;98 %, and 14, respectively. <br>
<br>
Third, a systematic study for the development of high-capacity immunoaffinity adsorbents was conducted. MAb, Fab, scFv and multimeric scFv fusions were investigated as affinity ligands. The accessibility of the binding sites had substantial effect on the binding capacity. It turned out to be largely impacted by the ligand format, ligand density, and orientation of ligands. Altogether, highest binding capacity was achieved for adsorbents with site-directed monomeric and multimeric scFv ligands; up to 20 mg/mL for a target protein of 50 kDa.}},
  author       = {{Sakhnini, Laila}},
  isbn         = {{978-91-7422-701-7}},
  keywords     = {{Monoclonal Antibodies; Aggregation; Biophysical Characterisation; Process Development; Affinity Adsorbents; Monoclonal Antibodies; Aggregation; Biophysical Characterisation; Process Development; Affinity Adsorbents}},
  language     = {{eng}},
  month        = {{10}},
  publisher    = {{Lund University, Faculty of Engineering}},
  school       = {{Lund University}},
  title        = {{Antibody-Fragments: Protein Engineering, Process Development, and their Application as Affinity Ligands}},
  url          = {{https://lup.lub.lu.se/search/files/71274001/L.Sakhnini_PhD_thesis.pdf}},
  year         = {{2019}},
}