Pilot-scale integrated continuous biomanufacturing for monoclonal antibodies including mild pH

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Toolbox for efficient development of high cell density perfusion process, integrated with continuous DSP

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Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells

Recombinant protein production can cause severe stress on cellular metabolism, resulting in limited titer and product quality. To investigate cellular and metabolic characteristics associated with these limitations, we compare HEK293 clones producing either erythropoietin (EPO) (secretory) or GFP (non-secretory) protein at different rates. Transcriptomic and functional analyses indicate significantly higher metabolism and oxidative phosphorylation in EPO producers compared with parental and GFP cells. In addition, ribosomal genes exhibit specific expression patterns depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased EPO secretion, we detect higher gene expression related to negative regulation of endoplasmic reticulum (ER) stress, including upregulation of ATF6B, which aids EPO production in a subset of clones by overexpression or small interfering RNA (siRNA) knockdown. Our results offer potential target pathways and genes for further development of the secretory power in mammalian cell factories

The human secretome

The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immuno-assays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood

Bifunctional ADAPTs: Opportunity for serological Half-life extension and Targeted therapy [Elektronisk resurs]

Small engineered scaffold proteins (ESPs) gain more and more popularity as biological drugs, due to their specificity and applicability in diagnostics and therapy. Thanks to their high stability, low immunogenicity and low production cost, they present themselves as a promising alternative to the market-leading antibodies. However, their relatively small size poses the risk of fast blood clearance, a circumstance advantageous for imaging purposes but a disadvantage in a therapeutic setting.This thesis has focused on introducing bifunctionality, the ability of the same engineered scaffold protein to exert more than one function, by applying different engineering approaches. Therefore, a new combinatorial protein library based on ABD-derived affinity proteins (ADAPTs) was generated, originating from a bacterial albumin-binding domain. From this library, it was possible to achieve protein modules with the ability to simultaneously bind to its intended target as well as to human serum albumin (HSA), a feature that has been shown to increase the binder’s half-life in the body. Specific binding modules were achieved by performing phage display selections towards the targets Tumor necrosis factor alpha (TNF⍺) and Interleukin-17c (IL-17c), both proinflammatory cytokines involved in many different inflammatory diseases and therefore interesting targets for therapeutic applications. The selection output was analyzed through sequencing and promising candidates were cloned and produced in Escherichia coli (E. coli), followed by a detailed characterization of each candidate including target binding, stability and their oligomeric state using methods like Surface Plasmon Resonance (SPR), Circular Dichroism (CD) and Size Exclusion Chromatography (SEC). It was possible to generate binders that passed all characterization criteria, most importantly showing simultaneous bispecificity to either TNF⍺ or IL-17c in combination with albumin. Each binder was then examined for their usefulness as a real therapeutic by successfully evaluating its ability to block the interaction of the cytokine and its specific receptor in vitro. These newly developed protein binders, showing high affinity towards their targets as well as keeping their initial binding to HSA, present another possibility to combine the advantages of small engineered scaffold proteins with those of typical larger proteins, allowing for more convenient production in bacteria leading to lower production costs and making them ideal candidates for future therapeutics.Furthermore, a previously developed ADAPT targeting the human epidermal growth factor receptor 2 (HER2) was genetically fused to an improved Horseradish Peroxidase (HRP) variant, thereby combining the idea of tumor-targeted therapy through the ADAPT with the utilization of HRP to enzymatically catalyze the prodrug IAA into its active form. After proving these new fusion proteins have similar binding kinetics to the target, as well as comparable enzymatic activities, as their free counterpart, the cytotoxic effects were put to the test in vitro. Hereby, the variants showed to benefit immensely through the addition of an ADAPT by being selectively effective only on HER2-positive cells. The evident advantage of these fusion proteins and their competency to be functionally produced in E. coli as well as the possibility to avoid an additional step of conjugation or coupling of affinity proteins to cytotoxic payloads, makes this approach a promising alternative for current procedures and another reason why ESPs are on the rise.Scaffoldproteiner, proteindomäner som är mindre till storleken, växer i popularitet som biologiska läkemedel tack vare deras specificitet och applicerbarhet inom både diagnostik och terapi. Detta, i kombination med deras stabilitet, låga immunogenicitet och låga produktionskostnad gör dem till lovande alternativ till nuvarande marknadsledande antikropparna. Att de är mindre till storleken medför dock en potentiell risk för kortare cirkulationstid i blodomloppet. Som egenskap kan detta ändå vara till fördel i diagnostisk visualisering, men till nackdel för terapeutiska ändamål.Denna avhandling har kretsat kring att utveckla dessa proteindomäner med målet att öka deras funktionalitet och därför skapades ett nytt kombinatoriskt proteinbibliotek baserat på ADAPT (ABD-derived affinity protein), som härstammar från en bakteriell albuminbindande domän. Från detta bibliotek var det möjligt att generera affinitetsproteiner som innehar förmågan att simultant binda till avsedda målprotein samt till humant serumalbumin (HSA), en egenskap som har visats öka bindarens halveringstid i kroppen. Specifika proteindomäner identifierades genom att utföra selektioner med fagdisplay mot tumörnekrosfaktor alfa (TNFa) och interleukin 17c (IL-17c); proinflammatoriska cytokiner involverade i ett stort antal olika inflammatoriska sjukdomar vilket gör dem intressanta som mål inom terapeutiska tillämpningar. Erhållna selektionskandidater har analyserats genom sekvensering och varianter som ansågs lovande klonades och producerades i Escherichia coli (E. coli), följt av en detaljerad utvärdering av varje kandidats individuella bindning till deras specifika målprotein, karakterisering av deras stabilitet såväl som deras oligomera tillstånd med hjälp av metoderna Surface Plasmon Resonance (SPR), Circular Dichroism (CD) och Size Exclusion Chromatography (SEC). Det var möjligt att generera bindare som uppfyllde samtliga karakteriseringskriterier, och som påvisade simultan bindning till TNFa eller IL-17c i kombination med albumin. Vidare undersöktes varje bindares förmåga att blockera respektive cytokins interaktion med dess specifika receptor in vitro för att utvärdera deras potentiella användbarhet som läkemedel. Dessa nyutvecklade proteinbindare, som uppvisar hög affinitet till sitt målprotein samt bibehåller sin naturliga bindning till HSA, utgör en ny möjlighet att kunna kombinera fördelarna hos mindre proteindomäner med de fördelar som typiskt tillhör större proteiner. Detta möjliggör en behändig och kostnadseffektiv produktion i bakterier vilket gör dem till ideala kandidater för framtida läkemedel.Vidare har en tidigare utvecklad ADAPT med specifik bindning till målproteinet HER2 (human epidermal growth factor receptor 2) genetiskt fusionerats med en förbättrad variant av enzymet HRP (horseradish peroxidase). Fusionsproteinet kombinerar målsökande tumörterapi genom ADAPT med användningen av HRP för att enzymatisk katalysera det inaktiva substratet IAA till dess aktiva form som cellgift. Efter att ha demonstrerat att dessa nya fusionsproteiner har liknande bindningskinetik till målproteinet samt jämförbar enzymatisk aktivitet som HRP allena, testades de cytotoxiska effekterna in vitro. Eftersom fusionsproteinerna endast verkade mot HER2-positiva celler påvisades fördelen med en genetisk fusion av enzymet till en HER2-bindande ADAPT. Denna tydliga fördel hos fusionsproteiner, samt att de kan produceras som funktionella enheter direkt i E. coli, innebär att det extra steget att konjugera eller koppla cytotoxiska substanser till affinitetsproteinet kan undvikas. Sammantaget påvisar resultaten att mindre proteindomäner, så som scaffoldproteiner, kan modifieras till att bli bifunktionella och är därför ett lovande framtida alternativ till nuvarande läkemedelsstrategier.</p

Addressing the Protease Bias in Quantitative Proteomics

Protein quantification strategies using multiple proteases have been shown to deliver poor interprotease accuracy in label-free mass spectrometry experiments. By utilizing six different proteases with different cleavage sites, this study explores the protease bias and its effect on accuracy and precision by using recombinant protein standards. We established 557 SRM assays, using a recombinant protein standard resource, toward 10 proteins in human plasma and determined their concentration with multiple proteases. The quantified peptides of these plasma proteins spanned 3 orders of magnitude (0.02-70 μM). In total, 60 peptides were used for absolute quantification and the majority of the peptides showed high robustness. The retained reproducibility was achieved by quantifying plasma proteins using spiked stable isotope standard recombinant proteins in a targeted proteomics workflow. </p

Targeted HER2-positive cancer therapy using ADAPT6 fused to horseradish peroxidase

Targeted cancer therapy is a promising alternative to the currently established cancer treatments, aiming to selectively kill cancer cells while sparing healthy tissues. Hereby, molecular targeting agents, such as monoclonal antibodies, are used to bind to cancer cell surface markers specifically. Although these agents have shown great clinical success, limitations still remain such as low tumor penetration and off-target effects. To overcome this limitation, novel fusion proteins comprised of the two proteins ADAPT6 and Horseradish Peroxidase (HRP) were engineered. Cancer cell targeting is hereby enabled by the small scaffold protein ADAPT6, engineered to specifically bind to human epidermal growth factor receptor 2 (HER2), a cell surface marker overexpressed in various cancer types, while the enzyme HRP oxidizes the nontoxic prodrug indole-3-acetic acid (IAA) which leads to the formation of free radicals and thereby to cytotoxic effects on cancer cells. The high affinity to HER2, as well as the enzymatic activity of HRP, were still present for the ADAPT6-HRP fusion proteins. Further, in vitro cytotoxicity assay using HER2-positive SKOV-3 cells revealed a clear advantage of the fusion proteins over free HRP by association of the fusion proteins directly to the cancer cells and therefore sustained cell killing. This novel strategy of combining ADAPT6 and HRP represents a promising approach and a viable alternative to antibody conjugation for targeted cancer therapy

Targeted proteomics analysis of plasma proteins using recombinant protein standards for addition only workflows

Targeted proteomics is an attractive approach for the analysis of blood proteins. Here, we describe a novel analytical platform based on isotope-labeled recombinant protein standards stored in a chaotropic agent and subsequently dried down to allow storage at ambient temperature. This enables a straightforward protocol suitable for robotic workstations. Plasma samples to be analyzed are simply added to the dried pellet followed by enzymatic treatment and mass spectrometry analysis. Here, we show that this approach can be used to precisely (coefficient of variation &lt;10%) determine the absolute concentrations in human plasma of hundred clinically relevant protein targets, spanning four orders of magnitude, using simultaneous analysis of 292 peptides. The use of this next-generation analytical platform for high-throughput clinical proteome profiling is discussed. </jats:p