Browsing by Subject "karakterisointi"

Sort by: Order: Results:

Now showing items 1-3 of 3
  • Takala, Hanna-Elina (Helsingin yliopisto, 2020)
    Monoclonal antibodies (mAbs) are widely used in the treatment of several diseases such as cancer and autoimmune diseases. Due to their high prices and growing consumption, therapeutic mAbs have become potential targets of falsification. This generates a demand for quick and efficient analytical procedures for identifying and characterizing mAbs in a case of suspected falsification. The structure of therapeutic mAbs consists of human or murine IgG framework, where unique complementarity determining regions (CDRs) are engineered with different recombinant techniques. Given the complex nature of the mAbs, they must be identified using multiple complementary analytical methods. Ten full-sized therapeutic mAbs, Fab-fragment abciximab and CTLA4-Fc-fusion protein belatacept were studied in order to find analytical methods for efficient characterization and identification. All studied antibodies were characterized by their charge and molecular weight by isoelectric focusing (IEF) in polyacrylamide gels, native and reduced SDS-PAGE, and size exclusion chromatography (SEC). Six mAbs, abciximab and belatacept were digested with trypsin, and the cleaved peptides were further analysed by RPLC-MS. In addition, quantification methods including SEC peak area measurements and Bradford protein assay were performed for all antibodies. As expected, SDS-PAGE of non-reduced and reduced mAbs gave little distinction between the mAbs. Both methods were however shown to be useful in the identification of the mAb nature, as they confirmed the presence of heavy chains, light chains, and disulfide bonds. IEF showed potential in mAb identification, as clear, partly distinguished patterns of charge variants were obtained. However, some improvements to the pH gradient are needed to enable better separation and pI estimation of basic variants. Determination of molecular size with SEC was found to be difficult, as there seemed to be no consistency between the calculated molecular weights based on measured elution times, and the theoretical molecular weights. Nevertheless, SEC brings added value in mAb quantification and detection of protein aggregation and fragmentation. Finally, RPLC-MS analysis of tryptic peptides resulted in mAb identification, with the measured sequence coverage of 87-97 %. Identification process may be enhanced by focusing on the known CDR-peptides prior the constant frame peptides. Given the structural similarity of therapeutic mAbs, identification of an unknown mAb requires combination of multiple analytical methods. If available, the use of reference mAb product obtained from a reliable source is recommended, as the identification may be based on comparative analyses using simpler analytical steps, e.g. IEF, SDS-PAGE and SEC. If no reference product is available, identification of the mAb requires peptide mapping and determination of the CRD sequences by RPLC-MS analysis. Further research is needed to find a suitable set of analytical methods for identification of all therapeutic mAbs.
  • Kataja, Sara (Helsingin yliopisto, 2018)
    Production of biofuels from non-food-based materials, such as lignocellulose, provides a good alternative for the traditional burning of fossil fuels. Some of the researched and existing biofuel applications are based on the utilization of enzymes. There are multiple cellulolytic enzymes required in the efficient hydrolysis of lignocellulose, and one of the key enzyme group is β-glucosidases. These enzymatic systems are mainly adopted from wood-decaying fungi. The overall enzymatic system consists of different types of cellulases that first degrade the crystalline cellulose to oligosaccharides and cellobiose. In the final step, β-glucosidases hydrolyse the oligosaccharides to glucose (a fermentable sugar). In fact, β-glucosidases are one of the limiting enzyme classes in this process, due to phenomena such as end-product inhibition. β-Glucosidases belong to Glycoside hydrolases (GH), that can be classified into different protein families. In an industrial perspective, the main interest resides in GH1 and GH3 family enzymes. Many industrially relevant extracellular β-glucosidases belong to GH3 family. However, intracellular GH1 β-glucosidases often exhibit higher tolerance to harsh conditions such as high substrate and product concentrations, high temperatures and low pH. The goal of this MSc thesis work was to purify and characterize a novel GH1 β-glucosidase, named NBG. Both GH1 and GH3 family enzymes were used as references for the characterization work. The GH3 reference enzyme was a β-glucosidase from Aspergillus niger (An Cel3A), derived from the commercial enzyme preparation Novozym 188. The used GH1 reference was a β-glucosidase from termite Nasutitermes takasagoensis (Nt GH1). The applicability of NBG β-glucosidase in biomass hydrolysis was also examined, together with possible considerations for applicability by other type of applications. The purification of An Cel3 reference enzyme was performed as described previously in literature. A novel protocol combining thermal treatment and low resolution IEX purification was developed for the NBG enzyme in this study. The enzyme’s activity on various pNP-substrates was determined, followed by pH stability, thermostability and inhibition studies. According to the result, NBG is a potential candidate for industrial use. The enzyme was found to be thermostable and active in a wide pH range when compared to the reference enzymes (stable up to 20 h at +60 ˚C and in pH 3.5 – 6.0). NBG also exhibited wider activity on pNP-substrates than the reference enzymes, highest specific activity being on pNPG, followed by moderate activity on pNPFuc and low activities on pNPGal and pNPXyl. Furthermore, NBG exhibited higher tolerance to inhibitors such as glucose and ethanol. Glucose inhibition was not observed until concentration of 200 mM for NBG, while in the same concentration the reference enzymes were almost completely inhibited. A Clear activation (of +16 %) by 100 mM glucose was observed with NBG. This enzyme also outperformed the An Cel3A-reference in ethanol tolerance, retaining activity better in 15 and 20 % ethanol. Activation by ethanol was also observed for both of the fungal enzymes, the most pronounced effect being observed for NBG in 15 % ethanol (+21 % of initial activity). The hydrolysis of insoluble cellulosic substrate (Avicel) was investigated using a commercial cellulase mixture (Celluclast 1.5L), where a semi-pure β-glucosidase preparation was added: novel β-glucosidase preparation (NBG (2-S2)) or the reference preparation An Cel3A (Nz188). According to the results, the NBG (2S-2) was outperformed by An Cel3A (Nz188) in Avicel 4 – 72 h hydrolysis experiments. The amount of reducing sugars released from Avicel was approximately 18–19 % higher with the commercial Nz188 preparation when compared to the 2S-2 preparation. Further analyses of samples revealed accumulation of cello-oligosacchardes, which may accumulate due to two possible reasons: Either the NBG enzyme does not possess high enough cellobiase activity (needed in biomass hydrolysis to glucose), or accumulation of cellobiose is due to transglycosylation activity of NBG. According to activity (and 3D modelling) data, NBG may not be a true β-glucosidase belonging to the EC (and having cellobiase activity). Further investigation of the possible substrate specificity and transglycosylation activity of the NBG will be needed in assessing its applicability in other types of biotechnical applications.
  • Valkonen, Sami (Helsingfors universitet, 2014)
    Microvesicles (MVs) are lipid bilayered membranous vesicles containing functional lipids, proteins, RNA and DNA that are produced by most cells. The physiological significance of MVs has become evident, and increased MV counts and the contents of MVs are nowadays also associated with different pathophysiological phenomena. The goal of the field is to use MVs as diagnostic and therapeutic tools. To achieve this, the understanding of the mechanisms of the functions of MVs should be understood better and additionally, reliable methods for the quantification and characterization of MVs should be developed and standardized. The aim of the study was to determine differences in platelet-derived MVs produced by different activation mechanisms. The second aim was to set up and optimize a protocol based on the reaction of sulphur, phosphate and vanillin (SPV) for measuring lipid content of MVs. The third aim was to study the effect of thrombin and proteinase inhibitor PPACK to the vesiculation of platelets. Platelets were isolated from the whole blood of healthy volunteers and vesicles were produced by platelet agonists mediating thrombogenic activation (thrombin and collagen, TC), pathophysiological activation (lipopolysaccharide, LPS) and Ca-ionophore (A23187) as positive control for vesiculation. Quantification and size determination of produced MVs was done using Nanoparticle Tracking Analysis (NTA). MVs were characterized by protein content using bicinchonic acid assay (BCA) and by lipid content using SPV-reaction. MVs had great activation-dependent differences in the lipid and the protein content. Activation with Ca-ionophore produced the most MVs, but the lipid and protein content was only a fraction from (patho)physiologically induced MVs. Only TC increased vesiculation. Vesicle subpopulations had significant difference in lipid content. Thrombin and proteinase inhibitor PPACK mediated inhibition of platelet formation in all of the activations, but the effect was not statistically significant. The mechanism of inhibition was likely to be proteinase inhibitor mediated. The isolation of vesicle populations using differential centrifugation proved to isolate studied populations only partially and the quantification method with NTA was susceptible to concentrated samples. SPV protocol reacted with different intensity to different lipids. In the future, quantification and isolation methods for MVs and the subpopulations of MVs should be improved. Additionally, to understand the physiologically relevant mechanisms of platelet-derived vesicle formation, the inhibitor experiments with PPACK should be continued, because the number of replicates was too low to see significant effects due to a large donor-dependent deviation. Since MVs are heterogenous cellular multitools affecting varying (patho)physiological phenomena, optimization and standardization of methods should be continued in order to study MVs properly.