Standard purification processes in large scale antibody production are largely dependent on Protein A chromatography. Protein A binds specifically to many subclasses of IgG with high affinity. However, in order to elute the proteins, the pH needs to be lowered. Since lowering of the pH can be detrimental to some antibodies, a milder purification process is of great interest. A variant of Protein A, called ZCa, has previously been engineered to bind to IgG in a calcium-dependent manner. The antibody binds to ZCa when calcium is present and releases when calcium is removed. For the IgG1 subclass, the elution still requires a slight lowering of the pH, which is why there is room for improvement of the molecule. A phage display selection has been performed with the aim to obtain calcium-dependent IgG1 binders that are able to release the antibodies upon calcium depletion at neutral pH. In addition, an attempt on increasing the alkaline stability of the binders was made. Sequence analysis of the selection output showed almost no indications of increased alkaline stability. Instead, M13K07 helper phages were exposed to new selections for increased alkaline tolerance which might be useful in future phage display selections. Even though the binders selected for in this project did show some promising characteristics, none of them were able to elute upon calcium depletion at neutral pH as aimed for. However, one of the variants did show promising results during most of the performed characterizations. Most interestingly, the elution properties of this variant could indicate a higher calcium-dependence in the interaction with the target than that of ZCa, although further characterizations need to be performed in order to draw any conclusions about possible improvements of this protein domain.

Vid Klinisk kemi och farmakologi, Specialkemi, vid Skånes universitetssjukhus, Lund, utförs kvantifiering med LC-MS/MS av antipsykotiska och antidepressiva läkemedelskoncentrationer i serum med acetonitril (ACN) som mobilfas. ACN är på grund av sin höga elueringsstyrka ett av de vanligaste organiska lösningsmedlen vid reversed phase (RP) kromatografi, men uppvisar samtidigt en hög toxicitet med risk för stora leveransproblem. I syfte att reducera mängden ACN undersöktes därför möjligheten till ett mobilfasbyte till metanol (MeOH). Ordinarie metod jämfördes med tre nyutvecklade metoder med MeOH-baserad mobilfas. I en av metoderna ändrades endast mobilfas och elueringsgradient, medan två av metoderna även använde andra sorters RP-kolonner med anpassade elueringsgradienter. Samtliga analyter uppvisade godkänd separation och retention vid eluering med MeOH, men stor fluktuation från referensmetod sågs vid kvantifieringen av flera analyter, däribland olanzapin, desmetylolaznapin och mirtazapin. Liknande avvikelser med avseende på regression och kvantifieringsdifferens observerades vid eluering med andra sorters RP-kolonner. Detta indikerar att vidare optimering av andra vätskekromatografiska och masspektrometriska parametrar bör utföras innan metoderna kan valideras. / The quantification of antipsychotics and antidepressants in human serum with LC-MS/MS is usually performed with acetonitrile (ACN) as mobile phase. ACN is one of the most common organic solvents in reversed phase (RP) chromatography thanks to its high elution strength but is also highly toxic and can at times suffer from major delivery problems. The present study investigated the possibility of replacing ACN with methanol (MeOH) as primary organic solvent with the purpose of reducing the total ACN-usage. Three newly developed methods for chromatographic analysis of antipsychotic and antidepressant drugs using MeOH as organic solvent as part of the mobile phase were compared to the routine method in regard to analyte separation and retention, as well as the relative quantification of substance. In one of the methods only the mobile phase and elution gradient was changed whereas different types of RP columns were applied in addition to the changed mobile phase in the other two. All substances showed acceptable separation and retention when eluted with MeOH but displayed large fluctuations in the quantification of the analyzed substances, more specifically olanzapine, desmethylolanzapine and mirtazapine, in comparison to the reference method. Similar deviations in terms of regression and quantification bias were observed when analytes were eluted with MeOH through other types of RP columns. This indicates that further optimization of other parameters relating to the chromatography and mass spectrometer should be performed before validation.

In the chemistry branch of science is often a chromatograph usedto separate and purify substances in a solution. In achromatograph, different detectors are used to analyse the samplesconstituents. Some detectors destroy the sample during analysis.Because it is undesirable to destroy the entire flow of sample, asmall mass is transferred to a second flow by a flow splitter. Inthis report, four principles to divert a small mass from one flowto another are developed and evaluated. The basic principles todivert the flows is tested and principal mock-ups are designed andmanufactured. A brief survey of the market is conducted and aproblem related to flow spitting is investigated. The problem isthe influence on yield through a chromatograph due to flowretaining. Designing and testing of different retaining systems isalso included in this report. All four initial principles proved to be plausible splittingtechniques. However, only two principles appeared to be feasiblefor direct implementation in devices comparable withchromatographs. One of the less feasible principle is covered byseveral patents. The other is difficult to manufacture in order tomeet the strict requirements associated with e.g. chromatographs.The testing of different retaining systems showed that smallertube inner diameter and how the tube is winded can reduce theretaining system influence on the yield significantly. The splitting techniques in this report are all feasible splittingtechniques, and the report can be used as a solid foundation fordevelopment future laboratory instruments. / Inom kemin förekommer ofta kromatografer för att rena och separera kemiska substanser iett prov. De separerade substanserna kan detekteras och analyseras av olika detektorer, därvissa detektorer förstör provet i analysprocessen. Då det oftast är önskvärt att bevara såmycket som möjligt av ett prov, för vidare studier, avleds därför en liten mängd av deseparerade substanserna till detektorn då en förstörande detektor används. I denna studieutvecklades och undersöktes fyra principer för att avleda mycket små volymer från ett flödetill ett annat. Flödesdelning och sammanförning med de grundläggande flödesdelningsprincipernatestades, principiella modeller konstruerades och tillverkades. Enmarknadsundersökning samt en undersökning av ett flödesdelningsassocierat probleminkluderas också i rapporten. Problemet består i minskning av utbyte på grund avbandbreddning i flödesfördröjningssystem. Bandbreddning studerades således i olika formerav flödesfördröjningssystem. Alla fyra principer visade sig vara möjliga flödesdelningstekniker varav två av sådan karaktäratt de skulle kunna realiseras i ett instrument såsom en kromatograf. Av de två andra omfattasden ena av flertalet patent och den andra skulle vara tillverkningstekniks svår att förverkligamed vedertagna tillverkningstekniker. Tester på olika typer avflödesfördröjningssystemutformningar visade att slangdimension och hur slangen är lindadhar stor inverkan på bandbreddning. Det återstår en hel del konceptuell utveckling då principerna bara behandlats pågrundläggande nivå. Genom de principiella modellerna ges en bild av hur principerna skullekunna förverkligas, men vidare ställningstaganden måste tas till bland annat materialval samtmonitorering och anpassning av flöden. Rapporten ger en god grund för framtidaproduktutveckling.