Whey is a by-product of cheese manufacture, containing a mixture of proteins of commercial value, each having unique attributes for nutritional, biological and food ingredient applications. A tremendous amount of whey, normally treated as a waste product, is produced worldwide each year. This work describes the cation-exchange adsorption of the two major whey proteins, alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) with the purpose of optimising a process for isolating them from whey. Adsorption of pure BLG and ALA was studied onto SP Sepharose FF using 0.1M acetate buffer. Batch experiments were carried out at various pH values for ALA and BLG, and the relevant Langmuir isotherm parameters, dissociation constant, Kd, and maximum binding capacity, qm, were determined. The optimum pH for separation was chosen to be pH 3.7. At pH 3.7, both Kd and qm pertaining to ALA were found to have higher numerical values than those of BLG, implying different characteristics of adsorption of the two proteins on this adsorbent. The Kd for the former protein was almost four times larger than the latter, while qm was 1.3 times higher. Packed-bed column adsorption was performed using a 1-ml column at pH 3.7, flow rate 1 ml/min and initial concentration of 3 mg/ml for BLG and 1.5 mg/ml for ALA both in 0.1M sodium acetate buffer. The t1/2 for the resulting ALA breakthrough was 75% longer than its BLG counterpart. The above results suggest the possibility of the occurrence of competitive adsorption between the proteins when adsorbed simultaneously. In traditional batch uptake experiments, the kinetic rate constants of ALA and BLG in both the single- and two-component systems were determined using the simple kinetic model. The values so obtained implied that BLG was adsorbed faster than ALA. In the confocal laser scanning microscopy experiments, the different behaviour of ALA and BLG in the single-component system with regard to their penetration within the adsorbent beads suggested that the two proteins have different transport mechanisms governing their adsorption. The two-component system results showed that ALA was able to displace BLG in spite of the lower affinity of the former protein to the adsorbent. The packed-bed adsorption and elution of a mixture of ALA and BLG were then investigated under the above conditions but using a 5-ml column. BLG breakthrough occurred first, and its concentration in the outlet exceeded its feed value by 1.6 fold before declining to the feed value, followed by the breakthrough of ALA. ALA displaced and eluted all the BLG from the column in a pure form. Pure ALA could then be eluted with good recovery. The single- and two-component breakthrough curves for ALA and BLG were simulated by the simple kinetic model using the isotherm parameters, but the overshoot phenomenon could only be predicted after correcting these parameters. The evidence of the competitive nature of adsorption observed in binary mixtures was used to develop a facile separation procedure for the two proteins from aqueous solutions of whey concentrate powders. A novel consecutive two-stage separation process was developed to separate ALA and BLG from whey concentrate mixtures. Almost all the BLG in the feed was recovered, with 78% being recovered at 95% purity and a further 20% at 86% purity. In addition, 67% of ALA was recovered, 48% at 54% purity and 19% at 60% purity. The correction factors employed for the pure binary mixture were used to simulate the breakthrough curves of the two proteins in experiments conducted with whey concentrate in each of the two stages of the novel separation process, and there was agreement between the experimental and theoretical results.

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京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23135号 / 薬科博第134号 / 新制||薬科||15(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 石濱 泰, 教授 松﨑 勝巳, 教授 加藤 博章 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Extracellular vesicles (EV) originate from various cell types and reflect the contents of the originating cells. EVs are ubiquitous in nearly all body fluids, including blood plasma, and exhibit significant potential as biomarkers in disease diagnostics. However, isolating EVs from blood plasma remains challenging due to the lack of a standardised method. This study aimed to compare and optimize a density gradient ultracentrifugation workflow (DUC) against size exclusion chromatography-cation exchange chromatography (SEC-CEC) and evaluate SEC versus SEC-CEC. Common contaminants during isolation include lipoproteins (LP); previous studies have shown that lowering the pH of blood plasma can precipitate LP, enhancing isolation efficiency. Acidified blood plasma was compared with neutral plasma for EV isolation using all above mentioned methods. To assess the ability of the isolation methods to purify contaminants while retaining maximal EV yield, samples were analysed using multiple techniques, including particle quantity, free proteins, LP-associated apolipoprotein B, purity index (μg protein/particle), and EV-associated surface markers. The results indicate potential for DUC, but further optimization is necessary to improve the method and its isolation of EV. SEC-CEC emerged as an effective method, reducing contaminants by 71% (SEC) to 99% (SEC-CEC), increasing purity by 80%, and yielding positive signals from EV markers (SEC-CEC). The effect of acidification was ambiguous, it reduced apolipoprotein-B levels in plasma pre-isolation. However, post- isolation, neutral plasma exhibited significantly lower contaminations, albeit at the expense of total particle content and risking EV loss. The study underscored several advantages of SEC-CEC but indicated that acidification did not optimise isolation efficiency.

Several novel polymer monoliths for the analysis of peptides and proteins were synthesized using polyethylene glycol diacrylate (PEGDA) as crosslinker. Photo-initiated copolymerization of polyethylene glycol methyl ether acrylate and PEGDA yielded an inert monolith that could be used for size exclusion liquid chromatography of peptides and proteins. This macroscopically uniform monolith did not shrink or swell in either water or tetrahydrofuran. More importantly, it was found to resist adsorption of both acidic and basic proteins in aqueous buffer without any organic solvent additives. A strong cation-exchange polymer monolith was synthesized by copolymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and PEGDA. A ternary porogen (water, methanol and ethyl ether) was found suitable to prepare a flow-through monolith with moderate pressure drop in aqueous buffer. The resulting monolith showed excellent ion exchange capillary liquid chromatography of peptides using a simple salt gradient. Extremely narrow peaks were obtained for the analysis of synthetic peptides, natural peptides and a protein digest. A peak capacity of 179 was achieved. Although the poly(AMPS) monolith demonstrated extraordinary performance, one main drawback of this monolith was its relatively strong hydrophobicity. A decrease in hydrophobicity was achieved by using more hydrophilic monomers (e.g., sulfoethyl methacrylate or vinyl sulfonic acid). The most hydrophilic poly(vinyl sulfonic acid) monolith provided high resolution cation-exchange liquid chromatography of protein standards and lipoproteins. Use of the new PEGDA biocompatible crosslinker over the conventional ethylene glycol dimethacrylate crosslinker for the preparation of polymer monoliths was found to be advantageous for the analysis of biological compounds in several chromatography modes.