| 1. |
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| 2. |
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| 3. |
- Ahlqvist, Josefin, et al.
(författare)
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Monitoring the production of inclusion bodies during fermentation and enzyme-linked immunosorbent assay analysis of intact inclusion bodies using cryogel minicolumn plates
- 2006
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 354:2, s. 229-237
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Tidskriftsartikel (refereegranskat)abstract
- A novel minicolumn chromatgraphic method to monitor the production of inclusion bodies during fermentation and anenzyme-linked immunosorbent assay (ELISA) system allowing direct analysis of the particles with surface-displayed antigens are described. A 33-kDa protein containing 306 amino acids with three sulfur bridges produced its inclusion bodies wits labeled with polyclonal antibodies against 15 amino acid (anti-A15) and 17 amino acid (anti-B17) residues at the N- and C-terminal ends of the protein, respectively. Labeled particles were bound to macroporous Monolithic protein A-cryogel adsorbents inserted into the open-ended wells of a 96-well plate (referred to as protein A-cryogel minicolumn plate). The concept behind this application is that the binding degree of inclusion bodies from lysed fermentation broth to the cryogel minicolumns increases with an increase in their concentration during fermentation. The technique allowed LIS to monitor the increase in the production levels of the inclusion bodies as the fermentation process progressed. The system also has a built-in quality parameter to ensure that the target protein has been fully expressed. Alternatively, inclusion bodies immobilized on phenyl-cryogel minicolumn plate were used in indirect ELISA based on anti-A15 and anti-B17 antibodies against terminal amino acid residues displayed oil the surface of inclusion bodies. Drainage-protected properties of the cryogel minicolumns allow performance of successive reactions with tested immunoglobulin G (IgG) samples and enzyme-conjugated secondary I-G and of enzymatic reaction within the adsorbent. (c) 2006 Elsevier Inc. All rights reserved.
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| 4. |
- Dainiak, Maria, et al.
(författare)
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Affinity cryogel monoliths for screening for optimal separation conditions and chromatographic separation of cells
- 2006
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1123:2, s. 145-150
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Tidskriftsartikel (refereegranskat)abstract
- Suitable conditions for separating cells using a chromatographic procedure were evaluated in parallel chromatography on minicolumns. A 96-hole minicolumn plate filled with cryogel monoliths (18.8 mm x 7.1 mm O) with immobilized concanavalin A was used. Chromatographic columns (113 mm x 7.1 mm O) were used for chromatographic resolution of a mixture of Saccharomyces cerevisiae and Escherichia coli cells. Separation of a cell mixture containing equal amounts of cells of both types performed in a column format under the determined optimal conditions, resulted in a quantitative capture of applied S. cerevisiae cells, while E. coli passed through the column. Bound S. cerevisiae cells were released by flow-induced detachment and by compression of the adsorbent in the presence of 0.3 M methyl U-D-manno-pyranoside. The flowthrough and the eluted fractions were analyzed by plate counting and by registering metabolic activity of S. cerevisiae cells in the eluted fractions after capturing on ConA-cryogel monoliths in a 96-minicolumn plate format. The flowthrough fraction contained E. coli cells with nearly 100% purity, whereas the fraction eluted by compression of the adsorbent contained viable S. cerevisiae cells with 95% purity. Thus, an efficient chromatographic separation of cells was achieved using affinity cryogel column.
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| 5. |
- Dainiak, Maria B, et al.
(författare)
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Gelatin-fibrinogen cryogel dermal matrices for wound repair: Preparation, optimisation and in vitro study.
- 2010
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Ingår i: Biomaterials. - : Elsevier BV. - 1878-5905 .- 0142-9612. ; 31, s. 67-76
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Tidskriftsartikel (refereegranskat)abstract
- Macroporous sponge-like gelatin-fibrinogen (Gl-Fg) scaffolds cross-linked with different concentrations (0.05-0.5%) of glutaraldehyde (GA) were produced using cryogelation technology, which allows for the preparation of highly porous scaffolds without compromising their mechanical properties, and is a more cost-efficient process than freeze-drying. The produced Gl-Fg-GA(X) scaffolds had a uniform interconnected open porous structure with a porosity of up to 90-92% and a pore size distribution of 10-120mum. All of the obtained cryogels were elastic and mechanically stable, except for the Gl-Fg-GA(0.05) scaffolds. Swelling kinetics and degradation rate, but not the porous structure of the cryogels, were strongly dependent on the degree of cross-linking. A ten-fold increase in the degree of cross-linking resulted in an almost 80-fold decrease in the rate of degradation in a solution of protease. Cryogels were seeded with primary dermal fibroblasts and the densities observed on the surface, plus the expression levels of collagen types I and III observed 5 days post-seeding, were similar to those observed on a control dermal substitute material, Integra((R)). Fibroblast proliferation and migration within the scaffolds were relative to the GA content. Glucose consumption rate was 3-fold higher on Gl-Fg-GA(0.1) than on Gl-Fg-GA(0.5) cryogels 10 days post-seeding. An enhanced cell motility on cryogels with reducing GA crosslinking was obtained after long time culture. Particularly marked cell infiltration was seen in gels using 0.1% GA as a crosslinker. The scaffold started to disintegrate after 42 days of in vitro culturing. The described in vitro studies demonstrated good potential of Gl-Fg-GA(0.1) scaffolds as matrices for wound healing.
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| 6. |
- Dainiak, Maria B., et al.
(författare)
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Macroporous monolithic hydrogels in a 96-minicolumn plate format for cell surface-analysis and integrated binding/quantification of cells
- 2007
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Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 40:4, s. 688-695
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Tidskriftsartikel (refereegranskat)abstract
- Macroporous monolithic hydrogels (cryogel monoliths; rods 12.5 mm x 7.1 mm diameter) are elastic, sponge-like materials with large (10-100 mu m), interconnected pores. Phenyl- and IMAC-(Me(II)-iminodiacetic acid)-cryogel monoliths were inserted into the open-ended wells of a standard 96-well plate, forming a system of 96 drainage-protected minicolumns, and were used in a parallel assay of hydrophobicity and affinity to immobilized metal ions of wild type Escherichia coli cells, recombinant E. coli cells with poly-His peptide displayed on the cell surface, and Bacillus halodurans cells in different growth phases. Bound cells were eluted with standard eluents or were detached by mechanical compression of affinity cryogel monoliths in the case of strongly bound cells. The possibility to carry out high throughput viability assays of bound cells was demonstrated on an example of analysis of recombinant E. coli cells bound to Cu(II)-IDA-cryogel monoliths and of yeast cells bound to ConA-cryogel monoliths, where the metabolic activity of cells was measured using tetrazolium salt XTT and pH indicator, neutral red, respectively. The developed system can be used for the rapid optimization of chromatographic separation of cells and for detection of cells of interest from a large number of medical and food samples.
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| 7. |
- Dainiak, Maria, et al.
(författare)
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Biomimetic macroporous hydrogel scaffolds in a high-throughput screening format for cell-based assays.
- 2008
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 24:6, s. 1373-1383
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Tidskriftsartikel (refereegranskat)abstract
- Macroporous hydrogels (MHs) hold great promise as scaffolds in tissue engineering and cell-based assays. In this study, the possibility of combination of three-dimensional (3D) cell culture with a miniaturized screening format was demonstrated on human colon cancer HCT116, human acute myeloid leukemia KG-1 cells, and embryonic fibroblasts cultured on MHs (12.5 mm x 7.1 mm I.D.) in a 96-minicolumn plate format. MHs were prepared by cryogelation technique and functionalized by coating with type I collagen and by copolymerization with agmatine-based mimetic of cell adhesive peptide RGD (abRGDm). Cancer cells formed multicellular aggregates while fibroblasts formed adhesions on abRGDm-containing and collagen-MHs but not on plain MHs, as was demonstrated by scanning electron microscopy. HCT116 and KG-1 cells grown as aggregates were more resistant to the treatment with cis-diaminedichloroplatinum (II) (cisplatin) and cytosine 1-beta-D-arabinofuranoside (Ara-C), respectively, during the first 18-24 h of incubation, than single cells grown on unmodified MH. HCT116 cells grown as 2D cultures in conventional 96-well tissue culture plates were 1.5- to 3.5-fold more sensitive to the treatment with 70 microM cisplatin than cells in 3D cultures in functionalized MHs. Further development of the described experimental system including matching of a specific cell type with appropriate extracellular matrix (ECM) components and 3D cocultures on ECM-modified MHs may provide a realistic in vitro experimental model for high-throughput toxicity tests.
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| 8. |
- Dainiak, Maria, et al.
(författare)
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Cell chromatography: Separation of different microbial cells using IMAC supermacroporous monolithic columns
- 2005
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 21:2, s. 644-649
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Tidskriftsartikel (refereegranskat)abstract
- Supermacroporous monolithic columns with CU2+-IDA ligands have been successfully used for chromatographic separation of different types of microbial cells. The bed of monolithic matrix is formed by a cryogel of poly(acrylamide) cross-linked with methylenebis(acrylamide) and has a network of large (10- 100 mu m) interconnected pores allowing unhindered passage of whole cells through the plain cryogel column containing no ligands. Two model systems have been studied: the mixtures of wild-type Escherichia coli (w.t. E. coli) and recombinant E. coli cells displaying poly-His peptides (His-tagged E. coli) and of w.t. E. coli and Bacillus halodurans cells. Wild-type E. coli and His-tagged E. coli were quantitatively captured from the feedstock containing equal amounts of both cell types and recovered by selective elution with imidazole and EDTA, with yields of 80% and 77%, respectively. The peak obtained after EDTA elution was 8-fold enriched with His-tagged E. coli cells as compared with the peak from imidazole elution, which contained mainly weakly bound w.t. E. coli cells. Haloalkalophilic B. halodurans cells had low affinity to the CU2+-IDA cryogel column and could be efficiently separated from a mixture with w.t. E. coli cells, which were retained and recovered in high yields from the column with imidazole gradient. All the cells maintained their viability after the chromatographic procedure. The results show that chromatography on affinity supermacroporous monolithic columns is a promising approach to efficient separations of individual cell types.
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| 9. |
- Dainiak, Maria, et al.
(författare)
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Chromatography of living cells using supermacroporous hydrogels, cryogels
- 2007
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Ingår i: Advances in Biochemical Engineering, Biotechnology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0724-6145. ; 106, s. 101-127
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Tidskriftsartikel (refereegranskat)abstract
- The preparative cell separation is an intrinsic requirement of various diagnostic, biotechnological and biomedical applications. Affinity chromatography is a promising technique for cell separation and is based on the interaction between a cell surface receptor and an immobilised ligand. Most of the currently available matrices have pore size smaller than the size of the cells and are not suitable for cell chromatography due to column clogging. Another problem encountered in chromatographic separation of cells is a difficulty to elute bound cells from affinity surfaces. Application of novel adsorbents, supermacroporous monolithic cryogels, allows overcoming these problems. Cryogels are characterised by highly interconnected large (10-100 mu m) pores, sponge-like morphology and high elasticity. They are easily derivatised with any ligand of choice. Convective migration can be used to transport the cells through the matrix. Target cells bind to affinity ligands, while other cells pass through the cryogel column non-retained and are removed during a washing step. Because of the spongy and elastic nature of the cryogel matrices, the cells are efficiently desorbed by mechanical compression of cryogels, which provides high cell viability and yields. The release of affinity bound cells by mechanical compression of a cryogel monolithic adsorbent is a unique and efficient way of cell detachment. This detachment strategy and the continuous macroporous structure make cryogels very attractive for application in cell separation chromatography.
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| 10. |
- Dainiak, Maria, et al.
(författare)
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Detachment of affinity-captured bioparticles by elastic deformation of a macroporous hydrogel
- 2006
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 103:4, s. 849-854
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Tidskriftsartikel (refereegranskat)abstract
- Adsorption of bioparticles to affinity surfaces involves polyvalent interactions, complicating greatly the recovery of the adsorbed material. A unique system for the efficient binding and release of different cells and particles is described. Affinity-bound bioparticles and synthetic particles are detached from the macroporous hydrogel matrix, a so-called cryogel, when the cryogel undergoes elastic deformation. The particle detachment upon elastic deformation is believed to be due to breaking of many of the multipoint attachments between the particles and the affinity matrix and the change in the distance between affinity ligands when the matrix is deformed. However, no release of affinity-bound protein occurred upon elastic deformation. The phenomenon of particle detachment upon elastic deformation is believed to be of a generic nature, because it was demonstrated for a variety of bioparticles of different sizes and for synthetic particles, for different ligand-receptor pairs (IgG-protein A, sugar-ConA, metal ion-chelating ligand), and when the deformation was caused by either external forces (mechanical deformation) or internal forces (the shrinkage of thermosensitive, macroporous hydrogel upon an increase in temperature). The elasticity of cryogel monoliths ensures high recovery of captured cells under mild conditions, with highly retained viability. This property, along with their continuous porous structure makes cryogel monoliths very attractive for applications in affinity cell separation.
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| 11. |
- Dainiak, Maria, et al.
(författare)
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Direct capture of product from fermentation broth using a cell-repelling ion exchanger.
- 2002
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 942:1-2, s. 123-131
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Tidskriftsartikel (refereegranskat)abstract
- A new technique for treating anion exchangers has been proposed allowing direct capture of the fermentation product, shikimic acid directly from the cell-containing fermentation broth. A layer of hydrophilic polymer, poly(acrylic acid) (PAA) has been physically adsorbed on the anion exchanger followed by a covalent cross-linking of PAA. The PAA layer is penetrable for small molecules despite being negatively charged as PAA is, but the polymer layer repels large negatively charged structures like cell debris and cells preventing them from adsorption to the chromatographic matrix. The binding capacity for pure shikimic was about 81 mg/ml adsorbent for both cross-linked PAA-Amberlite and native Amberlite in the fluidized mode of column operation. Binding capacity dropped to 17 and 15 mg per ml adsorbent, respectively, when using filtrated fermentation broth and to about 10 mg/ml adsorbent for cross-linked PAA-Amberlite when using directly the fermentation broth containing cells. Native Amberlite cannot be used for the direct capture of shikimic acid due to the immediate clogging of the column and the collapse of the expanded bed. The cross-linked PAA-Amberlite was used repeatedly for the direct adsorption of shikimic acid from the industrial fermentation broth.
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| 12. |
- Dainiak, Maria, et al.
(författare)
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Improved methods for prepurification and detection of Staphylococcal Enterotoxin B from cell-free culture filtrate
- 2005
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 21:4, s. 1347-1351
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Tidskriftsartikel (refereegranskat)abstract
- An improved ELISA method for the detection of Staphylococcal. Enterotoxin B (SEB) in protein A preparations is presented. Fab fragments were obtained by digestion with papain of anti-SEB IgG bound to SEB immobilized on Sepharose 4B. Anti-SEB and peroxidase-labeled Fab fragments from secondary antibodies were successfully used in a modified ELISA of SEB in protein A preparations. SEB-Sepharose was used repeatedly for the production of anti-SEB Fab fragments by papain digestion without loss of affinity. In addition, for the purification of SEB from crude culture filtrates, an initial step utilizing a combined heat and pH treatment for the removal of significant amounts of contaminating proteins without losses of toxin activity is presented. This pretreatment step yielded positive effects in further downstream processing considering both shortened time and an increase in total recovery.
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| 13. |
- Dainiak, Maria, et al.
(författare)
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Integrated isolation of antibody fragments from microbial cell culture fluids using supermacroporous cryogels
- 2004
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1045:1-2, s. 93-98
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Tidskriftsartikel (refereegranskat)abstract
- The present paper describes a chromatographic capture/purification step for the recovery of proteins directly from undiluted and unclarified cell culture broths using supermacroporous dimethylacrylamide (DMAA) cryogel. The interconnected character and the size (10-100 mum) of the pores of the adsorbent make it possible to process whole cell fermentation broths without blocking the column. Cu2+-iminodiacetic acid (IDA) DMAA cryogel has been used for the isolation and purification of excreted (His)(6)-tagged single chain (sc) Fv antibody fragments, (His)(6)-scFv, from E. coli cell culture. Bound protein was recovered with 0.2 M imidazole or with 20 mM EDTA and was practically cell-free. Chromatographic capture using Cu2+-IDA cryogel column was performed at flow rates of 300 and 600 cm/h, respectively and resulted in 84-96% recovery of (His)(6)-scFv fragments with a purification factor of 13-15. The DMAA cryogel adsorbent is mechanically stable, can withstand harsh cleaning-in-place procedure and is relatively inexpensive. Chromatographic isolation of proteins using cryogels allows efficient removal of cells and can be operated at a flow rate as high as 600 cm/h. This novel technique has proven to be a scalable process, does not require special equipment and can be a good alternative to expanded bed adsorption and other integrated isolation techniques. (C) 2004 Published by Elsevier B.V.
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| 14. |
- Dainiak, Maria, et al.
(författare)
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Methods in cell separations
- 2007
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Ingår i: Advances in Biochemical Engineering, Biotechnology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0724-6145. ; 106, s. 1-18
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Tidskriftsartikel (refereegranskat)abstract
- Research in the field of cell biology and biomedicine relies on technologies that fractionate cell populations and isolate rare cell types to high purity. A brief overview of methods and commercially available products currently used in cell separations is presented. Cell fractionation by size and density and highly selective affinity-based technologies such as affinity chromatography, fluorescence-activated cell sorting (FACS) and magnetic cell sorting are discussed in terms of throughput, yield, and purity.
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| 15. |
- Dainiak, Maria, et al.
(författare)
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Polyelectrolyte-coated ion exchangers for cell-resistant expanded bed adsorption
- 2002
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 18:4, s. 815-820
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Tidskriftsartikel (refereegranskat)abstract
- Adsorption chromatography in expanded beds is a widely used technology for direct capture of target proteins from fermentation broths. However, in many cases this method cannot be applied as a result of the strong tendency of cells or cell debris to interact with the adsorbent beads. To prevent contamination of the expanded bed with the biomass, STREAMLINE DEAE, anion exchanger designed for expanded bed adsorption, was modified with a layer of poly(acrylic acid) (PAA). The shielding layer of polyelectrolyte was attached to the surface of the matrix beads via electrostatic interactions. PAA with a high degree of polymerization was chosen to prevent diffusion of large polymer molecules into the pores of adsorbent. Thus, the shielding layer of PAA was adsorbed, only at the mouth of the pores of STREAMLINE DEAE beads and only marginally decreased the binding capacity of the ion exchanger for bovine serum albumin, the model protein in this study. PAA-coated STREAMLINE DEAE practically did not interact with yeast cells, which other-wise bound,strongly to the native adsorbent at neutral conditions. Cell-resistant PAA-coated anion exchanger was successfully used for isolation of BSA from the model protein mixture containing BSA, lysozyme (positively charged at applied conditions), and yeast cells. The layer of PAA was stable under mild elution conditions, and the modified adsorbent could be used in the repeated purification cycles.
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| 16. |
- Dainiak, Maria
(författare)
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Polyelectrolytes and Polycomplexes in Biotechnology
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Polyelectrolytes and polyelectrolyte complexes (PECs) are characterized by many useful properties that endow them with high potential in different areas of biotechnology. This thesis describes the development and analyses of some new systems and methods utilizing poly(acrylic acid) (PAA), poly(methacrylic acid) (PMA), poly (N-ethyl-4-vinylpyridinium bromide) (PEVP) and PMA-PEVP complexes. The model of chaperone action when the misfolded inactive forms of polypetides are removed from the medium preventing their aggregation was developed using PEVP and conjugates of PMA with monoclonal antibodies (IgG) specific to inactivated glyceraldehyde-3-phosphate dehydrogenase (iGAPDH). PMA-IgG conjugates “recognized” and bound iGAPDH in the solution of inactivated enzyme. Addition of stoichiometric amount of PEVP resulted in quantitative precipitation of the formed complex PMA-IgG-iGAPDH-PEVP which was removed by centrifugation. As the next step in the model development iGAPDH was covalently coupled to PMA. pH-dependent properties of IgG- and iGAPDH-PMA conjugates have been studied using turbidimetry and light scattering. The immunocomplexes iGAPDH / IgG-PMA and iGAPDH-PMA / IgG-PMA were shown to precipitate in acidic media. The competition of free iGAPDH and iGAPDH-PMA for binding with IgG-PMA and the dynamic release of refolded GAPDH, which looses affinity to IgG-PMA, into solution could be used for simulating chaperone action. Nonstoichiometric PEC formed by PMA and PEVP udergoes reversible precipitation from aqueous solution at any desired pH-value in the range 4.5-6.5 depending on the ionic strength and PEVP/PMA ratio in the complex. Antigen, iGAPDH, was covalently coupled to PEVP and the resulting conjugate was used for affinity precipitation of IgG using nonstoichiometric PEC. On the base of the developed affinity precipitation procedure a new method of the production of monovalent Fab fragments of IgG has been developed. Proteolysis of IgG in the presence of iGAPDH-PEVP conjugate followed by i) precipitation induced by PMA addition and pH shift from 7.3 to 6.5 and ii) elution at pH 4.0 resulted in 90 % immunologically competent Fab fragments. PAA was electrostatically attached to the surface of STREAMLINE DEAE beads in order to prevent binding of cells to the resin in expanded bed chromatography. The modified matrix was resistant towards interactions with the biomass and was successfully used for isolation of BSA from the model mixture containing bovine serum albumin, lysozyme (positively charged at applied conditions) and yeast cells. A new technique for coating strong anion exchangers with PAA layer has been developed. A layer of PAA was attached to the surface of Amberlite beads via electrostatic interactions followed by a covalent cross-linking of PAA. The cross-linked-PAA-Amberlite was used repeatedly for the direct adsorption of shikimic acid from the industrial fermentation broth. Native Amberlite could not be used due to the immediate clogging of the column with the biomass and the collapse of the expanded bed.
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| 17. |
- Galaev, Igor, et al.
(författare)
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Effect of matrix elasticity on affinity binding and release of bioparticles. Elution of bound cells by temperature-induced shrinkage of the smart macroporous hydrogel
- 2007
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 23:1, s. 35-40
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Tidskriftsartikel (refereegranskat)abstract
- The first step of bacterial or viral invasion is affinity and presumably multisite binding of bioparticles to an elastic matrix like a living tissue. We have demonstrated that model bioparticles such as inclusion bodies (spheres of about 1 mu m in size) Escherichia coli cells (rods 1 x 3 mu m), yeast cells (8 mu m spheres), and synthetic microgel particles (0.4 mu m spheres) are binding via different affinity interactions (IgG antibody-protein A, sugar-lectin, and metal ion-chelate) to a macroporous hydrogel (MH) matrix bearing appropriate ligands. The elastic deformation of the MH results in the detachment of affinity bound bioparticles. The particle detachment on elastic deformation is believed to be due to multipoint attachment of the particles to affinity matrix and the disturbance of the distance between affinity ligands when the matrix is deformed. No release of affinity bound protein occurred on elastic deformation. The efficiency of the particle release by the elastic deformation depends on the density of the ligands at the particle surface as well as on the elasticity of the matrix for relatively large particles. The release of the particles occurred irrespectively of whether the deformation was caused by external forces (mechanical deformation) or internal forces (the shrinkage of thermosensitive macroporous poly-N-isopropylacrylamide hydrogel on increase in temperature).
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| 18. |
- Galaev, Igor, et al.
(författare)
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High throughput processing of particulate-containing samples using supermacroporous elastic monoliths in microtiter (multiwell) plate format
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1065:2, s. 169-175
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Tidskriftsartikel (refereegranskat)abstract
- Two steps in parallel processing of multiple biosamples, namely, sample clarification and capture of the target protein, were integrated and combined with the direct assay of captured protein using a newly developed microtiter (96-well) plate system based on the monoliths of hydrophilic elastic supermacroporous material, cryogel. Cryogel monoliths have pore size large enough for microbial and mammalian cells to pass through unretained. Moreover, cryogel monoliths are elastic allowing them to be slightly compressed and easily introduced into the wells. When expanded, cryogel monoliths fill the well tightly with no risk of leakage in between the monolith and the walls of the well. The capillary forces keep the liquid inside the pores of the cryogel monolith making the monolith columns drainage protected. The application of a certain volume of liquid on top of a cryogel monolith column results in the displacement of exactly the same volume of liquid from the column. The concept of using supermacroporous gels in 96-well plate format offers new possibilities to the biotechnologist allowing separation of particulate matter, capturing of soluble material from particle containing media, and parallel assay of large number of non-clarified samples. (C) 2005 Elsevier B.V. All rights reserved.
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| 19. |
- Hanora, Amro, et al.
(författare)
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Screening of peptide affinity tags using immobilised metal affinity chromatography in 96-well plate format
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1087:1-2, s. 38-44
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Tidskriftsartikel (refereegranskat)abstract
- A method for high throughput screening of Green Fluorescent Proteins carrying metal binding tags in bacteria was developed. A random four amino acids tag-peptide library was successfully generated in E. coli. A 96-microtiter plate assembled with metal-iminodiacetic acid small cryogel columns was used for library screening. For the first time we were able to simultaneously screen a metal binding peptide tags library obtained from E. coli against different metal ions. From screening 25 different tags, three clones were able to bind to all metal ions studied (Ni2+, Zn2+, Co2+ and Cd2+). It was clearly demonstrated that the new construct could facilitate the screening of large peptide libraries. (c) 2005 Elsevier B.V. All rights reserved.
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| 20. |
- Kazakov, Sergey V., et al.
(författare)
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Light Scattering Study of the Antibody-Poly(methacrylic acid) and Antibody-Poly(acrylic acid) Conjugates in Aqueous Solutions
- 2001
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Ingår i: Macromolecular Bioscience. - 1616-5187. ; 1:4, s. 157-163
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Tidskriftsartikel (refereegranskat)abstract
- The effect of the conformational state of the polymer coil on the properties of protein-polymer conjugates has been studied for the conjugates of antibody (monoclonal antibody from 6C5 clone against inactivated rabbit muscle glyceraldehyde-3-phosphate dehydrogenase; Ab) with poly(methacrylic acid) (PMAA) or poly-(acrylic acid) (PAA). The pH-dependencies of molecular properties and structural parameters of aqueous solutions (radius of gyration, intensity of scattered light, hydrodynamic diameter, and polydisperisty index) of Ab, PMAA, and PAA, have been studied using static and dynamic light scattering techniques. While free Ab aggregates in solution and precipitates at its isoelectric point, the covalent attachment of a charged polymer to Ab prevents its association and shifts the precipitation point towards more acidic values (from pH 5.95 for Ab to pH ∼ 4.8 for Ab- PMAA). The predominant role of the conformational status of the polymer in the process of conjugate precipitation has been considered. Contrary to the precipitation of Ab-PMAA, the formation of stable colloidal particles was suggested for Ab-PAA at pH < 4.8. In the conjugates, polymer chains surround the protein globule in an extremely compact manner while Ab significantly affects the polymer conformation. The essentially larger hydrodynamic radii of conjugates, when compared with their radii of gyration, confirm the strong interaction of conjugates with solvent molecules.
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| 21. |
- Plieva, Fatima, et al.
(författare)
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Macroporous polyacrylamide monolithic gels with immobilized metal affinity ligands: The effect of porous structure and ligand coupling chemistry on protein binding
- 2006
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 1099-1352 .- 0952-3499. ; 19:4, s. 305-312
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Tidskriftsartikel (refereegranskat)abstract
- Macroporous polyacrylamide gels (MPAAG) with iminodiacetic acid (IDA) functionality were prepared by (i) chemical modification of polyacrylamide gel, (ii) co-polymerization of acrylamide with allyl glycidyl ether (AGE) and N,N' metylene-bis(acrylamide) (MBAAm) followed by coupling IDA ligand or (iii) by copolymerization of acrylamide and MBAAm with functional monomer carrying IDA-functionality (1-(N,N-bis(carboxymethyl)amino-3-allylglycerol). Screening for optimized conditions for the production of the MPAAG with required porous properties was performed in a 96-well chromatographic format that allowed parallel production and analysis of the MPAAG prepared from reaction mixtures with different compositions. Scanning electron microscopy of the fabricated MPAAG revealed two different types of the porous structures: monomodal macroporous structure with large interconnected pores separated by dense non-porous pore walls in case of plain gels or gels produced via copolymerization with AGE. The other type of the MPAAG (gel produced via co-polymerization with functional monomer carrying IDA-functionality) had bimodal pore structure with large interconnected pores separated by the pore walls pierced through with micropores. The effect of different modifications of MPAAG monoliths and of porous structure of the MPAAG (monomodal and bimodal porous structure) on protein binding has been evaluated. Copyright (c) 2006 John Wiley & Sons, Ltd.
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| 22. |
- 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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| 23. |
- 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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| 24. |
- 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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| 25. |
- 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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| 26. |
- Savina, Irina N., et al.
(författare)
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Biomimetic Macroporous Hydrogels: Protein Ligand Distribution and Cell Response to the Ligand Architecture in the Scaffold
- 2009
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Ingår i: Journal of Biomaterials Science. Polymer Edition. - 0920-5063. ; 20:12, s. 1781-1795
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Tidskriftsartikel (refereegranskat)abstract
- Macroporous hydrogels (MHs), cryogels, are a new type of biomaterials for tissue engineering that can be produced from any natural or synthetic polymer that forms a gel. Synthetic MHs are rendered bioactive by surface or bulk modifications with extracellular matrix components. In this study, cell response to the architecture of protein ligands, bovine type-I collagen (CG) and human fibrinogen (Fg), immobilised using different methods on poly(2-hydroxyethyl methacrylate) (pHEMA) macroporous hydrogels (MHs) was analysed. Bulk modification was performed by cross-linking cryo-co-polymerisation of HEMA and poly(ethylene glycol) diacrylate (PEGA) in the presence of proteins (CG/ pHEMA and Fg/pHEMA MHs). The polymer surface was modified by covalent immobilisation of the proteins to the active epoxy (ep) groups present on pHEMA after hydrogel fabrication (CG-epHEMA and Fg-epHEMA MHs). The concentration of proteins in protein/pHEMA and protein-epHEMA MHs was 80-85 and 130-140 mu g/ml hydrogel, respectively. It was demonstrated by immunostaining and confocal laser scanning microscopy that bulk modification resulted in spreading of CG in the polymer matrix and spot-like distribution of Fg. On the contrary, surface modification resulted in spot-like distribution of CG and uniform spreading of Fg, which evenly coated the surface. Proliferation rate of fibroblasts was higher on MHs with even distribution of the ligands, i.e., on Fg-epHEMA and CG/ pHEMA. After 30 days of growth, fibroblasts formed several monolayers and deposited extracellular matrix filling the pores of these MHs. The best result in terms of cell proliferation was obtained on Fg-epHEMA. The ligands displayed on surface of these scaffolds were in native conformation, while in bulk-modified CG/ pHEMA MHs most of the proteins were buried inside the polymer matrix and were less accessible for interactions with specific antibodies and cells. The method used for MH modification with bioligands strongly affects spatial distribution, density and conformation of the ligand on the scaffold surface, which, in turn, influence cell-surface interactions. The optimal type of modification varies depending on intrinsic properties of proteins and MHs. (C) Koninklijke Brill NV, Leiden, 2009
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