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

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 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.

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 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.

• author: Sakhnini, Laila
• supervisor: Leif Bülow; Anja K. Pedersen; Maria Dainiak
• opponent: Associate Professor van Alstine, James, Royal Institute of Technology, Stockholm.
• organization: Pure and Applied Biochemistry
• publishing date: 2019-10-23
• type: Thesis
• publication status: published
• subject: Medical Biotechnology; Bioprocess Technology
• keywords: Monoclonal Antibodies, Aggregation, Biophysical Characterisation, Process Development, Affinity Adsorbents, Monoclonal Antibodies, Aggregation, Biophysical Characterisation, Process Development, Affinity Adsorbents
• pages: 80 pages
• defense location: Lecture hall KC:B, Kemicentrum, Naturvetarvägen 14, Faculty of Engineering LTH, Lund
• defense date: 2019-11-18 13:00:00
• ISBN: 978-91-7422-700-0
• ISBN: 978-91-7422-701-7
• 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