| 201. |
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| 202. |
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| 203. |
- Sakhnini, Laila Ismail, et al.
(författare)
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Designing monoclonal antibody fragment-based affinity resins with high binding capacity by thiol-directed immobilisation and optimisation of pore/ligand size ratio
- 2016
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1468, s. 143-153
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Tidskriftsartikel (refereegranskat)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-, 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.
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| 204. |
- Sakhnini, Laila I, et al.
(författare)
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Improving the Developability of an Antigen Binding Fragment by Aspartate Substitutions
- 2019
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 58:24, s. 2750-2759
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Tidskriftsartikel (refereegranskat)abstract
- Aggregation can be a major challenge in the development of antibody-based pharmaceuticals as it can compromise the quality of the product during bioprocessing, formulation, and drug administration. To avoid aggregation, developability assessment is often run in parallel with functional optimization in the early screening phases to flag and deselect problematic molecules. As developability assessment can be demanding with regard to time and resources, there is a high focus on the development of molecule design strategies for engineering molecules with a high developability potential. Previously, Dudgeon et al. [(2012) Proc. Natl. Acad. Sci. U. S. A. 109, 10879-10884] demonstrated how Asp substitutions at specific positions in human variable domains and single-chain variable fragments could decrease the aggregation propensity. Here, we have investigated whether these Asp substitutions would improve the developability potential of a murine antigen binding fragment (Fab). A full combinatorial library consisting of 393 Fab variants with single, double, and triple Asp substitutions was first screened in silico with Rosetta; thereafter, 26 variants with the highest predicted thermodynamic stability were selected for production. All variants were subjected to a set of developability studies. Interestingly, most variants had thermodynamic stability on par with or improved relative to that of the wild type. Twenty-five of the variants exhibited improved nonspecificity. Half of the variants exhibited improved aggregation resistance. Strikingly, while we observed remarkable improvement in the developability potential, the Asp substitutions had no substantial effect on the antigenic binding affinity. Altogether, by combining the insertion of negative charges and the in silico screen based on computational models, we were able to improve the developability of the Fab rapidly.
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| 205. |
- Sakhnini, Laila I., et al.
(författare)
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Multimeric fusion single-chain variable fragments as potential novel high-capacity ligands
- 2020
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 10:4, s. 507-514
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Tidskriftsartikel (refereegranskat)abstract
- In basic and applied biotechnology, design of affinity ligands has become essential for high-capacity applications such as affinity-based downstream processes for therapeutic molecules. Here, we established a proof-of-concept for the use of multimeric fusion single-chain variable fragment (scFvs) as high-capacity ligands in affinity adsorbents. Mono- and di/tri-scFvs separated by Pro-rich negatively charged linkers were designed, produced, and immobilized to 6% cross-linked agarose beads. Frontal binding experiments with a target protein of 50 kDa resulted in up to 20 mg·mL−1 and 82% in dynamic binding capacity and utilization yield, respectively, at 100% breakthrough. The utilization of the binding sites was impacted by the ligand format and ligand density, rather than limitation in pore size of adsorbent as previously suggested. Overall, we demonstrated that multimeric fusion scFvs can successfully be developed and used as high-capacity ligands in affinity adsorbents, enabling lean process design and alignment with process specifications.
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| 206. |
- Sakhnini, Laila I., et al.
(författare)
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Optimizing selectivity of anion hydrophobic multimodal chromatography for purification of a single-chain variable fragment
- 2019
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Ingår i: Engineering in Life Sciences. - : Wiley. - 1618-0240 .- 1618-2863. ; 19:7, s. 490-501
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Tidskriftsartikel (refereegranskat)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 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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| 207. |
- Sandén, Anna Maria, et al.
(författare)
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Limiting factors in Escherichia coli fed-batch production of recombinant proteins
- 2003
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 81:2, s. 158-166
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Tidskriftsartikel (refereegranskat)abstract
- Fed-batch production of recombinant beta-galactosidase in E. coli was studied with respect to the specific growth rate at induction. The cultivations were designed to induce protein production by IPTG at a glucose feed rate corresponding to high (mu = 0.5 h(-1)) or low (mu = 0.1 h(-1)) specific growth rate. Protein production rate was approximately 100% higher at the higher specific growth rate, resulting in the accumulation of beta-galactosidase up to 30% of the total cell protein. Transcription analysis showed that beta-galactosidase-specific messenger RNA was immediately formed after induction (<5 min), but the amount was the same in both cases and was thus not the initial limiting factor. The content of ribosomes, as represented by rRNA, rapidly decreased with specific growth rate from a relative level of 100%, at the high specific growth rate, to 20% at the low specific growth rate. At high specific growth rate, ribosomes were additionally degraded upon induction due to the high production level. Translation therefore seemed to be the initial limiting factor of the protein synthesis capacity. The alarmone guanosine tetraphosphate increased at both high and low feed level inductions, indicating an induction-forced starvation of charged tRNA and/or glucose. The altered physiological status was also detected by the formation of acetic acid. However, the higher production rate resulted in high-level accumulation of acetic acid, which was absent at low feed rate production. Acetic acid production is thus coupled to the high product formation rate and is proposed to be due either to a precursor drain of Krebs cycle intermediates and a time lag before induction of the glyoxalate shunt, or to single amino acid overflow, since the model product is relatively poor in glycin and alanin. In conclusion, it is proposed that production at high specific growth rate becomes precursor-limited, while production at low specific growth rate is carbon- and/or energy-limited.
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| 208. |
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| 209. |
- Sandin, Leonard, et al.
(författare)
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Working with Nature-Based Solutions: Synthesis and mapping of status in the Nordics
- 2023
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- The world is currently facing a biodiversity and climate crisis which are globally interlinked. Nature-based solutions (NBS), defined as “actions to protect, sustainably manage, and restore natural and modified ecosystems that address societal challenges effectively and adaptively, simultaneously benefiting people and nature” is part of the solution to these challenges. Here we give a status overview of nature-based solutions in the Nordic countries, obtained within the S-ITUATION project[1] focusing on 1) what is the current status of research on NBS in the Nordic countries? 2) what policy framework(s) exist for NBS in the Nordic countries? 3) what challenges do Nordic countries experience in the process of mainstreaming NBS? 4) what key examples of projects implementing NBS exist in the Nordic countries? We have done this using several approaches: 1) a review of the academic literature, providing insights on the status of research on NBS in the Nordic countries; 2) a grey literature review in each Nordic country, to describe the policy framework for NBS and practical implementation of NBS projects across the Nordic countries; 3) compilation of a Nordic NBS case projects catalogue, which contains implemented case projects from each Nordic country, using NBS in all major ecosystems: terrestrial (forests and agricultural land), freshwater, coastal and marine, to show the breadth of NBS used in the Nordic countries, 4) Nordic NBS stakeholder consultations.Research on NBS across the Nordics includes several research initiatives. Currently the most central research initiatives are the Nordic Council of Ministers programme on NBS, which is a focused four-year programme. Many Nordic universities and research institutes are also involved in different research projects focusing on or including NBS and there is an exponential interest from researchers in this area. Most of these research projects are targeting NBS in urban areas. In a structured peer-review of scientific publications using the term ‘nature-based solutions’, 64 research papers were found related to the Nordic countries. These studies varied from large-scale ecosystem-based approaches to small-scale NBS. Most of the studies assessed the NBS functions in relation to biophysical qualities, such as water retention capacity, flood risk reduction, health benefits and biodiversity contribution, but there were also studies focusing on potential economic benefits from NBS. Regarding policy frameworks it is evident that these are at different stages of development when it comes to mainstreaming the concept of NBS into policy across the Nordics. Norway and Sweden have adopted the term to a larger degree than Denmark, Finland and Iceland. Still, all five countries conserve, restore and work actively on developing sustainable use of nature, but use other terms (e.g., ‘blue-green infrastructures or solutions’, ‘restoration’, or ‘ecosystem services’) in their policies and guidelines.NBS governance and implementation is an area that is currently advancing rapidly. At the same time, there are still several challenges as well as also opportunities for using NBS to mitigate and adapt to climate change, protect biodiversity and ensure human well-being. Regarding challenges and gaps, we divide these into 1) natural-scientific and technical knowledge gaps, 2) economic shortcomings, 3) regulatory, governance, and policy challenges, and 4) weak stakeholder collaboration. In the project we have identified 54 key examples of projects implementing NBS in the Nordic countries. Most of these cases were related to freshwater, followed by urban/artificial NBS. The number of implemented NBS projects has increased, especially in the last couple of years. Our key messages and recommendations for future mainstreaming of NBS are: 1) clear political prioritization is needed to mainstream NBS into policy and practice, 2) appropriate institutional structures, procedures and policy instruments at all governance levels are essential to facilitate the implementation of NBS, 3) better funding structures for NBS are needed, 4) we need to develop common standards, long-term monitoring and better cost-benefit evaluations of NBS, and 5) the knowledge base in all phases of NBS projects needs to be strengthened.
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| 210. |
- Scott, Robert A., et al.
(författare)
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Large-scale association analyses identify new loci influencing glycemic traits and provide insight into the underlying biological pathways
- 2012
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Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718. ; 44:9, s. 991-1005
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Tidskriftsartikel (refereegranskat)abstract
- Through genome-wide association meta-analyses of up to 133,010 individuals of European ancestry without diabetes, including individuals newly genotyped using the Metabochip, we have increased the number of confirmed loci influencing glycemic traits to 53, of which 33 also increase type 2 diabetes risk (q < 0.05). Loci influencing fasting insulin concentration showed association with lipid levels and fat distribution, suggesting impact on insulin resistance. Gene-based analyses identified further biologically plausible loci, suggesting that additional loci beyond those reaching genome-wide significance are likely to represent real associations. This conclusion is supported by an excess of directionally consistent and nominally significant signals between discovery and follow-up studies. Functional analysis of these newly discovered loci will further improve our understanding of glycemic control.
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