Hemofili A är en X-länkad ärftlig sjukdom som påverkar blodkoagulationen och beror på avsaknaden eller bristen på proteinet faktor VIII (fVIII). Nuwiq® är en rekombinant fVIII B- domän borttagen produkt från Octapharma AB som behandlar hemofili A och produceras med en cellinje av mänskliga njurceller från embryon. FVIII är 170 kDa som hel-längds protein, men klyvs till 90 kDa och 80 kDa kedjor innan de släpps ut i kroppen för cirkulation. Enligt reglementen måste 170 kDa kedjan vara under en specifik gräns under odlings processen för den farmaceutiska produkten och den relative distributionen av de tre olika isoformerna behövs mätas. Idag upparbetas proverna och analyseras med en droppkolon och reverse-phase high performance liquid chromatography (HPLC). Droppkolonen är en tidskrävande process och målet med detta projekt var att hitta en metod som kan förkorta tiden för detta analyssteg. I denna studie ska möjligheterna utvärderas för att använda Western Blot metod och att använda ett ÄKTA pure system för att rena proverna innan de analyseras med HPLC. Resultatet från Western Blot visa liknades resultat som HPLC analysen gör och en linjäritets, noggrannhets och precisions studie utfördes. Linjäritets studien resulterade i att provkoncentrationen bestämdes till 2 IU/mL. Noggrannhets testerna visade att det var liknande resultat men med mindre skillnader mellan de två metoderna och viss olikhet är förväntad då HPLC analyserar med avseende på hydrofobisitet och Western Blot använder antikroppar och kemiluminiscens. En mer omfattande precisions studie behövs innan några beslut kan tas angående en ändring av analysmetod. Western Blot visade lovande resultat och skulle förkorta metodtiden med ungefär 1-2 dagar. De första ÄKTA reningarna med odlingsprover visade inget protein i eluatet, men med en högre protein mängd pålagd på kolonen från ett prov med mindre cell rester hade eluatet en bra mängd protein i sig. Analysen av eluatet med Western Blot visade goda resultat. Fortsatta tester behövs göras med ÄKTA reningen för att metoden ska kunna betrakta det som en möjlig metod att använda på laboratoriet för att analysera faktor VIII protein. / Hemophilia A is a X-linked hereditary disease that effects the coagulation of the blood and is due to the absence or deficiency of the protein factor VIII (FVIII). Nuwiq® is a recombinant FVIII B-domain deleted product from Octapharma AB that treats hemophilia A and are produced with human embryonic kidney cell lines. The fVIII protein is 170 kDa full length but are cleaved into a 90 kDa chain and an 80 kDa chain before release into circulation in the body. Due to regulations the 170 kDa chain needs to be below a certain limit during the cultivation process of the pharmaceutical product and the relative distribution between the three isoforms needs to be measured. Today the sample is prepared and analysed through a drop column and reverse-phase high performance liquid chromatography (HPLC). The drop column is a time-consuming process and the aim for this project was to find a method that can reduce the time for this analysis step. This study investigated the possibilities to use Western Blot as a method and to use an ÄKTA pure system for the preparation of the samples and then analyse with HPLC. Results from Western Blot showed similar results as the HPLC analysis and a linearity, accuracy and precision study were performed. The linearity study showed promising results for a sample concentration of 2 IU/mL to be used. Accuracy testing showed similar but slightly different results between the two methods and some differences are expected since the HPLC analyses on hydrophobicity and Western Blot uses antibodies and chemiluminescence. There would need to be a more extensive precision study before any decision could be made about changing the analysis method. Western Blot showed promising results and would shorten the method time with around 1-2 days. The first ÄKTA purification runs with cultivation samples did not show any protein levels in the eluate, but with a higher protein amount put on the column from a sample with less cell debris the eluate had good amount of protein in it. The analysis of the eluate with Western Blot showed good results. Further testing of the ÄKTA purific tion is needed to consider it an option to use at the laboratory for analysis of factor VIII protein.

Transkription är stokastisk, där utbrottsmässiga episoder av RNA-transkription genererar RNA-molekyler. Trots att detta är en kärndel av eukaryotiskt liv, är lite känt om hur DNA-bindande transkriptionsfaktorer och transkriptionella kofaktorer formar gen-specifik transkriptionell utbrottskinetik. Syftet med detta examensarbete var att tyda rollerna hos kofaktorerna Med14 och P300/CBP inom transkriptionell utbrottskinetik. För detta ändamål användes Auxin inducible degron systemet för snabb nedbrytning av Med14 eller P300/CBP-proteiner i HCT116-celler, följt av Smart-seq3xpress single cell-RNA-sekvensering. Ett särskild fokus i denna avhandling var även att utvärdera förmågan att härleda direkta  genuttrycksförändringar genom analys av introniska reads – detta då introner ko-transkriptionellt splitsas och dess nyttjande skulle fånga effekter av mycket närliggande transkription. Resultaten visar en tidsberoende minskning av introniskt innehåll och en nedreglering av genuttryck för majoriteten av generna i de behandlade cellinjerna, medan opåverkade kontroller inte visar sådana trender. Utbrottskinetikresultaten indikerar att det inte finns någon korrelation mellan P300/CBP-pertuberade cellers geners ursprungliga utbrottsstorlek och några trender i genuttryckets relativa förändring, medan detsamma kan sägas för Med14-pertuberade cellers geners utbrottsfrekvens. Svaga trender från P300/CBP-påverkade cellers utbrottskinetik och uttrycksändring kan antyda att deras utbrottsfrekvens och inte utbrottsstorlek har påverkats. Resultaten antyder att perturbationen var framgångsrik och att P300/CBP inte påverkar utbrottsstorlek samt att Med14 kan reglera utbrottsfrekvensen för alla påverkade gener i lika hög grad. Vidare forskning behövs inom utbrottskinetikdata för att utöka vår förståelse av denna studies implikationer gällande Med14:s och P300/CBP:s reglerande roller på transkriptionella utbrott. / Transcription is stochastic with episodes of RNA transcription generating bursts of RNA molecules. Despite being a core part of eukaryotic life, little is known about how DNA-binding transcription factors and transcriptional co-factors shape gene-specific transcriptional bursting kinetics. The aim of this thesis was to decipher the roles of the co-factors Med14 and P300/CBP in transcriptional burst kinetics. To this end, the Auxin inducible degron system was used for rapid Med14 or P300/CBP protein degradation in HCT116 cells, followed by Smart-seq3xpress single-cell RNA-sequencing. A particular focus of this thesis was to evaluate the abilities to infer direct gene expression changes by analysis of intronic reads – since introns are co-transcriptionally spliced and would capture very recent transcription. Results show a time dependent decrease of intronic contents and a downregulation in gene expression for a majority of genes in the perturbed cell lines, while unperturbed controls show no such trends. Bursting kinetics results indicate that there is no correlation between P300/CBP perturbed cells’ gene’s original bursting size and any trends in gene expression fold change while the same can be said for Med14 perturbed cell’s gene’s burst frequency. Weak trends from P300/CBP perturbed cells’ bursting kinetics and expression fold change could imply that their bursting frequency and not bursting size has been affected. The results imply that the perturbation was successful and that P300/CBP does not affect bursting size as well as that Med14 could regulate bursting frequency for all affected genes to an equal degree. Further research is needed into the bursting kinetics data to expand our understanding of this study’s implications regarding regulatory roles of Med14 and P300/CBP on transcriptional bursting.

Aptamerer är nukleinsyra-baserade molekyler som binder specifikt till en målstruktur. Aptamerer har flera fördelar över antikroppar, så som snabb och billig framtagning och produktion. Multimera aptamer-baserade strukturer har visats ge bättre resultat än monomera aptamerer, men framtagningen av sådana strukturer är tidskrävande och icke-skalbar. Denna studie utforskar generering och selektion av multimera aptamer-baserade strukturer genom slumpmässig ligering och in vitro-evolution, med virusliknande partiklar av norovirus GII.2 och GII.4 som målstruktur. Optimering av ligeringsförhållanden visade att en större andel aptamerer, flerarmade fragment, och linjära fragment resulterade i störst diversitet i den ursprungliga strukturblandingen. Selektionsexperiment uppvisade kraftig positiv selektion för strukturer innehållande aptameren Buf-2, vilket indikerar att den har hög affinitet för båda genotyper. Aptameren SMV21 uppvisade också positiv selektion för båda genotyper. Studien finner också positiv selektion av multimera aptamer-baserade strukturer, vilket bekräftar att de binder starkare till sin målstruktur. Potentiella sekvenser med hög affinitet togs fram genom att generera konsensus-sekvenser från sekvenseringsdatan av de selekterade strukturerna. SPR användes för att mäta affiniteten av de selekterade strukturerna till norovirus, men på grund av ospecifik binding kunde inga slutsatser dras. / Aptamers are nucleic acid-based targeted binders that hold advantages over antibodies, such as cheap and fast development and production. Multimeric aptamer-based structures have shown improved performance compared to monomeric aptamers, but the development of such structures is time-consuming and unscalable. This study explores the generation and selection of multimeric aptamer-based structures through random ligation and in vitro evolution, targeting norovirus GII.2 and GII.4. Optimization of ligation conditions was performed, revealing that a bigger proportion of aptamers, multi-armed fragments, and linear fragments ensures a diverse initial structure pool. Selection experiments demonstrated a strong positive selection for structures containing the Buf-2 aptamer, indicating its high affinity for both norovirus genotypes. The SMV21 aptamer also showed positive selection for both genotypes. The study further found that multimeric aptamer-based structures experience positive selection, confirming their stronger binding to the desired target. Potential strong-binding sequences were obtained by generating consensus sequences from sequencing data of the selected strong binders. SPR was employed to determine the affinity of the selected binders to the norovirus, but the results were inconclusive due to unspecific binding.

Denna studie undersöker cellstorlekens inverkan på metabola processer, med särskilt fokus på skillnader mellan senescenta och icke-senescenta celler under varierande glukosförhållanden. Genom fluorescerande färgning, avbildning och flödescytometri analyserades nukleär och cellulär morfologi, DNA-innehåll, glukosupptag och cellvolym i cellinjerna HeLa Kyoto och DU-145. Resultaten visar signifikanta morfologiska förändringar i senescenta celler, inklusive ökad nukleär och cellulär storlek, högre aspektförhållanden och minskad nukleär konvexitet. Senescens associeras med minskat glukosupptag och förändrad metabolisk aktivitet, där större celler uppvisar lägre metabola hastigheter. Dessa fynd indikerar att cellulär senescens väsentligt påverkar metaboliska processer och morfologi, oberoende av glukoskoncentration. Forskningen förbättrar vår förståelse av cellulär metabolism och senescens, och erbjuder potentiella implikationer för terapeutiska strategier mot metabola störningar och cancer. Framtida studier bör undersöka de specifika mekanismerna bakom dessa förändringar och deras bredare tillämpningar inom medicinsk vetenskap.

Integrationen av artificiell intelligens (AI) i bioprocessapplikationer har framträtt som en transformerande metod inom bioteknik- och läkemedelsindustrin, och lovar betydande framsteg i effektivitet, verkan och hållbarhet. Genom att utnyttja avancerade algoritmer, såsom djupinlärning och förstärkningsinlärning, kan AI-system förutsäga och generera nya ligandstrukturer med hög affinitet för sina mål, såsom monoklonala antikroppar (mAbs) i detta projekt. Detta projekt syftar till att utvärdera potentialen hos AI-genererade affinitetsproteiner och validera de datorsimulerade förutsägelserna genom att undersöka bindningseffektiviteten och stabiliteten hos dessa ligander under verkliga förhållanden. Flera våtlabbstekniker användes för att uttrycka och rena de AI-designade proteinerna. Affinitetskromatografi var en teknik som användes för rening, följt av ytplasmonresonans (Biacore) för att studera interaktionen mellan de genererade affinitetsproteinerna och mAbs. Analyseresultat från SDS-PAGE och masspektrometri visade att de flesta proteiner kunde renas med hjälp av affinitetskromatografi. Emellertid visade karaktärisering med Biacore att de flesta proteiner inte interagerade med mAbs, förutom ett designat protein. Cirkulär dikroism (CD) spektrometri som användes för att visualisera sekundärstrukturen i proteiner visade att de flesta proteiner var veckade och bibehöll alfahelixar och betaflak jämfört med det vilda typen proteinet. Sammanfattningsvis ger denna forskning värdefulla insikter i utmaningarna vid utvärdering och karaktärisering av AI-genererade proteiner. Ytterligare forskningsinsatser bör fokusera på att förfina experimentella förhållanden och visualisera sekundärstrukturerna hos de genererade proteinerna för en djupare förståelse av deras stabilitetsproblem. / The integration of artificial intelligence (AI) into bioprocess applications has emerged as a transformative approach in the biotechnology and pharmaceutical industries, promising significant advancements in efficiency, efficacy, and sustainability. By leveraging advanced algorithms, such as deep learning and reinforcement learning, AI systems can predict and generate novel ligand structures with high affinity for their targets, such as monoclonal antibodies(mAbs) in this project. This project aims to evaluate the potential of AI-generated affinity protein ligands and validate the computational predictions by examining the binding efficiency and stability of these ligands in real-world conditions. Several wet lab techniques were employed to express and purify the AI-designed proteins. Affinity chromatography was one technique used for purification, followed by surface plasmon resonance (Biacore) to study the interaction between the generated affinity proteins and mAbs. Analysis results from SDS-PAGE and mass spectrometry showed that most proteins could be purified using affinity chromatography. However, characterization using Biacore revealed that most proteins did not interact with mAbs, except for one designed protein. Circular dichroism (CD) spectrometry used to visualize the secondary structure in proteins showed that most proteins were folded and retained alpha helices and beta sheets when compared to the wild type protein. In conclusion, this research provides valuable insights into the challenges in evaluating and characterizing AI-generated proteins. Further research efforts should focus on refining experimental conditions and visualizing the secondary structures of the generated proteins for a more in-depth understanding of their stability issues.

No description available.

High-throughput drug screening (HTS) in live cells is often a vital part of the preclinical anticancer drug discovery process. So far, two-dimensional (2D) monolayer cell cultures have been the most prevalent model in HTS endeavors. However, 2D cell cultures often fail to recapitulate the complex microenvironments of in vivo tumors. Monolayer cultures are highly proliferative and generally do not contain quiescent cells, thought to be one of the main reasons for the anticancer therapy failure in clinic. Thus, there is a need for in vitro cellular models that would increase predictive value of preclinical research results. The utilization of more complex three-dimensional (3D) cell cultures, such as multicellular tumor spheroids (MCTS), which contain both proliferating and quiescent cells, has therefore been proposed. However, difficult handling and high costs still pose significant hurdles for application of MCTS for HTS. In this work, we aimed to develop novel assays to apply MCTS for HTS and drug evaluation. We also set out to identify cellular processes that could be targeted to selectively eradicate quiescent cancer cells. In Paper I, we developed a novel MCTS-based HTS assay and found that nutrient-deprived and hypoxic cancer cells are selectively vulnerable to treatment with inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). We also identified nitazoxanide, an FDA-approved anthelmintic agent, to act as an OXPHOS inhibitor and to potentiate the effects of standard chemotherapy in vivo. Subsequently, in Paper II we applied the high-throughput gene-expression profiling method for MCTS-based drug screening. This led to discovery that quiescent cells up-regulate the mevalonate pathway upon OXPHOS inhibition and that the combination of OXPHOS inhibitors and mevalonate pathway inhibitors (statins) results in synergistic toxicity in this cell population. In Paper III, we developed a novel spheroid-based drug combination-screening platform and identified a set of molecules that synergize with nitazoxanide to eradicate quiescent cancer cells. Finally, in Paper IV, we applied our MCTS-based methods to evaluate the effects of phosphodiesterase (PDE) inhibitors in PDE3A-expressing cell lines. In summary, this work illustrates how MCTS-based HTS yields potential to reveal and exploit previously unrecognized tumor-specific vulnerabilities. It also underscores the importance of cell culture conditions in preclinical drug discovery endeavors.

Various molecular pathways in the brain shape our understanding of good and bad, as well as our motivation to seek and avoid such stimuli. This work evolves around how systemic inflammation causes aversion; and why general unpleasant states such as sickness, stress, pain and nausea are encoded by our brain as undesirable; and contrary to these questions, how drugs of abuse can subjugate the motivational neurocircuitry of the brain. A common feature of these various disease states is involvement of the motivational neurocircuitry - from mesolimbic to striatonigral pathways. Having an intact motivational system is what helps us evade negative outcomes and approach natural positive reinforcers, which is essential for our survival. During disease-states the motivational neurocircuitry may be overthrown by the molecular mechanisms that originally were meant to aid us. In study I, to investigate how inflammation is perceived as aversive, we used a behavioral test based on Pavlovian place conditioning with the aversive inflammatory stimulus E. coli lipopolysaccharide (LPS). Using a combination of cell-type specific gene deletions, pharmacology, and chemogenetics, we uncovered that systemic inflammation triggered aversion by MyD88-dependent activation of the brain endothelium followed by COX1-mediated cerebral prostaglandin E2 (PGE2) synthesis. Moreover, we showed that inflammation-induced PGE2 targeted EP1 receptors on striatal dopamine D1 receptor–expressing neurons and that this signaling sequence induced aversion through GABA-mediated inhibition of dopaminergic cells. Finally, inflammation-induced aversion was not an indirect consequence of fever or anorexia but constituted an independent inflammatory symptom triggered by a unique molecular mechanism. Collectively, these findings demonstrate that PGE2-mediated modulation of the dopaminergic circuitry is a key mechanism underlying inflammation-induced aversion. In study II, we investigate the role of peripheral IFN-γ in LPS induced conditioned place aversion by employing a strategy based on global and cell-type specific gene deletions, combined with measures of gene-expression. LPS induced IFN-ɣ expression in the blood, and deletion of IFN-ɣ or its receptor prevented conditioned place aversion (CPA) to LPS. LPS increased the expression of chemokine Cxcl10 in the striatum of normal mice. This induction was absent in mice lacking IFN-ɣ receptors or Myd88 in blood brain barrier endothelial cells. Furthermore, inflammation-induced aversion was blocked in mice lacking Cxcl10 or its receptor Cxcr3. Finally, mice with a selective deletion of the IFN-ɣ receptor in brain endothelial cells did not develop inflammation-induced aversion. Collectively, these findings demonstrate that circulating IFN-ɣ binding to receptors on brain endothelial cells which induces Cxcl10, is a central link in the signaling chain eliciting inflammation-induced aversion. In study III, we explored the role of melanocortin 4 receptors (MC4Rs) in aversive processing using genetically modified mice in CPA to various stimuli. In normal mice, robust aversions were induced by systemic inflammation, nausea, pain and kappa opioid receptor-induced dysphoria. In sharp contrast, mice lacking MC4Rs displayed preference towards most of the aversive stimuli, but were indifferent to pain. The unusual flip from aversion to reward in mice lacking MC4Rs was dopamine-dependent and associated with a change from decreased to increased activity of the dopamine system. The responses to aversive stimuli were normalized when MC4Rs were re-expressed on dopamine D1 receptor-expressing cells or in the striatum of mice otherwise lacking MC4Rs. Furthermore, activation of arcuate nucleus proopiomelanocortin neurons projecting to the ventral striatum increased the activity of striatal neurons in a MC4R-dependent manner and elicited aversion. Our findings demonstrate that melanocortin signaling through striatal MC4Rs is critical for assigning negative motivational valence to harmful stimuli. The neurotransmitter acetylcholine has been implied in reward learning and drug addiction. However, the role of cholinergic receptor subtypes in such processes remains elusive. In study IV we investigated the function of muscarinic M4Rs on dopamine D1R expressing neurons and acetylcholinergic neurons, using transgenic mice in various reward-enforced behaviors and in a “waiting”-impulsivity test. Mice lacking M4-receptors from D1-receptor expressing neurons exhibited an escalated reward seeking phenotype towards cocaine and natural reward, in Pavlovian conditioning and an operant self-administration task, respectively. In addition, the M4-D1RCre mice showed impaired waiting impulsivity in the 5-choice-serial-reaction-time-task. On the contrary, mice without M4Rs in acetylcholinergic neurons were unable to learn positive reinforcement to natural reward and cocaine, in an operant runway paradigm and in Pavlovian conditioning.  Immediate early gene expression mirrored the behavioral findings arising from M4R-D1R knockout, as cocaine induced cFos and FosB was significantly increased in the forebrain of M4-D1RCre mice, whereas it remained normal in the M4R-ChatCre mice. Our study illustrates that muscarinic M4Rs on specific neural populations, either cholinergic or D1R-expressing, are pivotal for learning processes related to both natural reward and drugs of abuse, with opposing functionality.

Thiopurine S-methyltransferase (TPMT) belongs to the Class I S-adenosylmethionine-dependent methyltransferase (SAM-MT) super family of structurally related proteins. Common to the members of this large protein family is the catalysis of methylation reactions using S-adenosylmethionine (SAM) as a methyl group donor, although SAM-MTs act on a wide range of different substrates and carry out numerous biologically important functions. While the natural function of TPMT is unknown, this enzyme is involved in the metabolism of thiopurines, a class of pharmaceutical substances administered in treatment of immune-related disorders. Specifically, methylation by TPMT inactivates thiopurines and their metabolic intermediates, which reduces the efficacy of clinical treatment and increases the risk of adverse side effects. To further complicate matters, TPMT is a polymorphic enzyme with over 40 naturally occurring variants known to date, most of which exhibit lowered methylation activity towards thiopurines. Consequently, there are individual variations in TPMTmediated thiopurine inactivation, and the administered dose has to be adjusted prior to clinical treatment to avoid harmful side effects. Although the clinical relevance of TPMT is well established, few studies have investigated the molecular causes of the reduced methylation activity of variant proteins. In this thesis, the results of biophysical characterization of two variant proteins, TPMT*6 (Y180F) and TPMT*8 (R215H), are presented. While the properties of TPMT*8 were indistinguishable from those of the wild-type protein, TPMT*6 was found to be somewhat destabilized. Interestingly, the TPMT*6 amino acid substitution did not affect the functionality or folding pattern of the variant protein. Therefore, the decreased in vivo functionality reported for TPMT*6 is probably caused by increased proteolytic degradation in response to the reduced stability of this protein variant, rather than loss of function. Also presented herein are novel methodological approaches for studies of TPMT and its variants. Firstly, the advantages of using 8-anilinonaphthalene-1-sulfonic acid (ANS) to probe TPMT tertiary structure and active site integrity are presented. ANS binds exclusively to the native state of TPMT with high affinity (KD ~ 0.2 μm) and a 1:1 ratio. The stability of TPMT was dramatically increased by binding of ANS, which was shown to co-localize with the structurally similar adenine moiety of the cofactor SAM. Secondly, an enzyme activity assay based on isothermal titration calorimetry (ITC) is presented. Using this approach, the kinetics of 6-MP and 6-TG methylation by TPMT has been characterized.

Antalet monoklonala antikroppar som används i läkemedel ökar kraftigt. Dessa läkemedel är dyra och risken för förfalskning är stor. Behovet att utveckla en metod för snabb och precis identifiering av monoklonala antikroppar är därför brådskande. För identifiering utfördes analyser med Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS), Capillary Gel Electrophoresis (CGE) and Capillary Zone Electrophoresis (CZE) på nio monoklonala antikroppar. Fokuset var att undersöka huruvida signifikanta fysiokemiska egenskaper och unika aminosyrasekvenser var närvarande och kunde urskiljas. Olika analyser med MALDI-ToF-MS användes till att både separera de monoklonala antikropparna baserat på dess fysiokemiska egenskaper, och annotera aminosyrasekvenser innehållande nyckelfragment. Med metoderna baserade på kapillärelektrofores uppnåddes också separation. CZE föredras framför CGE då mängden data som erhålls från CZE är större och provberedningen är enklare. Sammanfattningsvis utformades ett protokoll för identifieringsprocessen, vilket inleds med MALDI-ToF-MS-analyser av monoklonala antikroppar på reducerad form mot kända referenser. Därefter är en hypotes formulerad utifrån vilka antikroppar som ser mest lika ut. Slutligen analyseras dessa med CZE för fastställning av den monoklonala antikroppens identitet. / The number of monoclonal antibodies used in pharmaceuticals is increasing sharply. These medicines are expensive, and the risk of counterfeiting is high. The need to develop a method for rapid and precise identification of monoclonal antibodies is therefore urgent. For identification, analyses were performed with Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS), Capillary Gel Electrophoresis (CGE) and Capillary Zone Electrophoresis (CZE) on nine monoclonal antibodies. The focus was to investigate whether significant physiochemical features and unique amino acid sequences were present and could be distinguished. Various analyses with MALDI-ToF-MS were used to both separate the monoclonal antibodies based on their physicochemical properties and annotate amino acid sequences containing key fragments. With the methods based on capillary electrophoresis, separation was also achieved. CZE is preferred over CGE as the amount of data obtained from CZE is greater and sample preparation is simpler. In summary, an identification process protocol was designed and is initiated with MALDI-ToF-MS analyses of reduced-form monoclonal antibodies against known references. A hypothesis is then formulated based on which antibodies look the most similar. Finally, these are analysed by CZE to determine the identity of the monoclonal antibody.