Introduction. Plaque psoriasis (psoriasis vulgaris) is a chronic disease and the most common type of psoriasis. It is charactarized by well-defined areas with silvery scaling, erythema, puritus and sometimes pain. Psoriasis affects about 1.5 - 3 % of the world population. Patients with psoriasis often suffer with comorbidies which makes drug therapy essential in relieving symptoms. Mild to moderate disease is treated with topical therapy such as corticosteroids and retinoid creams and with phototherapy. More severe disease is treated with systemic therapy e.g. methotrexate, cyclopsorine and retinoids. Patients who do not respond well to these treatments can be put on antibody therapy, e.g., secukinumab. Secukinumab is a monoclonal antibody that specifically targets the IL-17A. It is used to treat moderate to severe psoriasis. Secukinumab binds to IL-17A and inhibits it to interact with IL-17R. This leads to downregulation of immune response and symptom relieving. Other monoclonal antibodies that are used are risankizumab that binds to the p19 subunit of IL-23 and ustekinumab that binds to the p40 subunit of IL-12 and IL-23. Clinical psoriasis symptoms are evaluated with the Psoriasis Area Severity Index (PASI) from 0 till 72 and with the Inverstigator’s Global Assessment (IGA) from 0 till 5. Method. This thesis is a literature review with an aim to evaluate the efficacy and safety of secukinumab in treating psoriasis vulgaris. The search for articles was done in PubMed with the search words ‘’secukinumab’’ and ‘’plaque psoriasis’’. Included articles were RCT-studies published between 2014 and 2022. Moreover, these studies used the PASI and the IGA scoring system. This thesis excluded studies with children. Overall, this thesis included 6 trials reported in 5 articles. Results. The trials ERASURE, FIXTURE and CAIN demonstrated that 300 mg and 150 mg secukinumab per day were effective in treating moderate to severe psoriasis vulgaris compared to  placebo and etarnecept. The trials CLARITY and CLEAR demonstrated that 300 mg secukinumab was effective in treating moderate to severe psoriasis vulgaris compared to ustekinumab. The trial IMMerge demonstrated that risankizumab was superior in treating psoriasis vulgaris compared to secukinumab. Most of the adverse effects were mild and moderate and the most common reported were nasopharyngitis, upper respiratory tract infection, diarrhea and headache.Conclusion. Secukinumab demonstrates good efficacy and safety in the treatment of moderate to severe psoriasis in patients who have not received a satisfactory result from other drugs therapies.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19379号 / 農博第2149号 / 新制||農||1037(附属図書館) / 学位論文||H28||N4959(農学部図書室) / 32393 / 新制||農||1037 / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 髙部 圭司, 教授 髙野 俊幸, 教授 杉山 淳司 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20281号 / 医博第4240号 / 新制||医||1021(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 岩田 想, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Monoclonal antibodies are large, complex biomolecules that can be difficult to characterize. Characterization is important because of the various post translational modifications that can occur during manufacturing, processing, and storage. Some modifications can lead to efficacy and safety issues and therefore are heavily monitored. A leading way to monitor various modifications is by using liquid chromatography. The high sensitivity, reproducibility, and ability to quantitate analytes makes it very attractive for monoclonal antibody characterization. The large molecular size of monoclonal antibodies (150 kDa) makes them challenging to separate efficiently and with high enough resolution to be helpful. New column technologies that would help improve protein separation efficiencies and slectivities would greatly help in this challenging process. In this thesis, three novel bonded phases are developed for the separation of monoclonal antibodies including a weak anion and cation exchanger (WAX, CEX) for the separation of charged species as well as a novel hydrophilic interaction chromatography (HILIC) for the separation of glycoforms. Column develop is achieved by optimizing selectivity and improving efficiency of separations by altering particle surface chemistry.

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Immunoglobulin (Ig) proteins, also known as antibodies, are important molecules with great variability in their amino acid sequences. Aberrantly overproduced monoclonal Ig light chain (LC) proteins may aggregate into a β-sheet featured structure, and deposit in the extracellular space; this pathologic process, called primary amyloidosis or Ig LC amyloidosis (AL) causes problems to multiple organs during the course of the disease. Post-translational modifications (PTMs), which remain to be explored, are likely an important factor affecting the formation of AL fibrils. In addition, therapeutic monoclonal antibodies (mAbs) are widely employed because of their high specificity and low side effects. Using plants as the expression platform is commercially attractive although this approach has been hampered by low protein expression yield and undesired glycosylation patterns. The investigations detailed in this dissertation focus on the analyses of Ig proteins derived from several human and plant sources. A method combining 2D SDS-PAGE separation and mass spectrometry (MS) analysis was used for de novo sequencing of Ig in a fat biopsy for which the corresponding DNA was unavailable, and for characterizing the LC proteins found in autopsied kidney, serum and urine samples from patients with AL amyloidosis whose LC-DNA was sequenced. The PTMs of each LC were extensively characterized with different enzymes and various tandem MS techniques including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD) and electron transfer dissociation (ECD). PTMs observed include truncations, mono-/di-chlorination of the tyrosine residues and a nitrile group formed from the primary amine on lysine side chains. All these may play critical roles in the fibrillogenesis and/or the disease pathogenesis. Experimental evidence supports the hypothesis that the proteolytic processing of amyloidogenic LCs occurs after deposition in the organ. Characterization of a plant-derived HSV8 mAb was accomplished using high-performance liquid chromatography separation and gel display followed by various MS methods. Three N-terminal and one C-terminal truncations were found. The N-glycan moiety attached to the heavy chain was also analyzed. The MS method established helps to elucidate important structural information on therapeutic mAbs in complex mixtures, potentially contributing to optimization of plant systems that may produce more stable mAbs.

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No description available.

The biopharmaceutical industry has experienced remarkable progress in the upstream production capacity of life-saving proteins. This is while the downstream processing has failed to keep pace, including unit operations which are working close to their physical limit with no economy of scale. Column chromatography which is an integral unit in different stages of downstream purification is considered as the major bottleneck in this section. The packed-bed resin media is costly and the processes are labor-intensive and extremely time consuming. Membrane chromatography which uses a stack of adsorptive membranes as the chromatographic media is one of the most promising alternatives for conventional chromatography techniques. The performance of membrane adsorbers is consistent over a wide range of flow rates which is owing to the dominance of convective solute transport as opposed to the diffusion-based nature of mass transfer within the pores of the resin beads. This translates to much higher productivity and considerably lower buffer consumption (even as high as 95%), leading to much lower overall processing costs. The other advantages are significantly lower footprints and decreased pressure drops, both contributing to diminished capital costs. Membrane adsorbers are greatly scalable and used in a single-use manner. The latter eliminates the cleaning and validation steps and brings about much shorter processing times and higher flexibility in process development. Due to the performance advantages of membrane chromatography, this technique is now widely used in purification of high volumes of samples in late-stage polishing. Currently available membrane adsorbers have radial-flow spiral-wound configuration with high frontal surface area to bed height ratio according to which dilute impurities are removed in a flow-through format at very high flow rates and low pressure drops. Nevertheless, they fail to give high-resolution for bind-and-elute separations which makes them unsuitable for many unit operations, highly restricting their application. Severe design deficiencies such as large dead volumes and varying membrane area over the bed height result in broad and poorly resolved peaks. Herein, a novel device design was successfully developed which addresses the abovementioned shortcomings. The laterally-fed membrane chromatography (LFMC) devices house a stack of rectangular membrane sheets with two rectangular lateral channels on both sides of the stack as the feed and permeate channels. The design offers balanced pressure over the sides of the stack as well as even solute flow path lengths due to which the solute residence time is very uniform. Also, the small dead volumes minimize the dispersion effects. These features make the LFMC technology highly suitable for bind-and-elute applications, the improvement which is brought about by a simple design. The devices are easy to fabricate and highly scalable. The LFMC devices containing cation-exchange (CEX) membranes with 7 mL bed volume were examined for bind-and-elute separation where they outperformed the equivalent commercially available radial-flow devices. The design was further modified to give even lower dead volumes and more cost-effective fabrication. The latest embodiment of the device gave resolutions which were comparable with the ones obtained with the commercially packed resin columns in 1 mL and 5 mL scale with consistency over wide range of flow rates. The results were all acquired using a three component model protein system. Upon the approval of suitability of the device for bind-and-elute separation, the CEX-LFMC was used for purification of monoclonal antibodies (mAbs), the largest class of biopharmaceuticals. The device showed great performance in separation of mAb charge variants when extensively shallow gradients (60 membrane bed volumes) were required. The devices offered very stable conductivity gradients at high flow rates. LFMC devices in three different preparative scales gave great performance in separation of mAb aggregates which was approved for different mAb samples. The other application studied with the CEX-LFMC devices was the single-step preparative purification of mono-PEGylated proteins which is as well very challenging due to the physicochemical similarities between the target molecules and the impurities. Collectively, the LFMC devices combine the high-resolution with high-productivity which is highly desirable in downstream purification of biological molecules with great potential to expand the application of membrane chromatography. Finally, the LFMC devices were modified to adapt the analytical scale where they were integrated with a stack of hydrophilized PVDF membranes. The device successfully delivered ultra-fast separation of mAb aggregates in less than 1.5 minutes based on hydrophobic interaction membrane chromatography (HIMC). The assay times achieved with the HI-LFMC technique outclassed the currently available ultra-high performance chromatography (UPLC) methods at the same time with being extremely cost-effective. The application of the LFMC technology in analytical scale has great potential to offer cheap and rapid analysis in process development and quality control section of biopharmaceutical manufacturing. / Thesis / Doctor of Philosophy (PhD)