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Mammalian Protein Expression and Characterization Tools for Next Generation Biologics

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Mammalian Protein Expression and Characterization Tools for Next Generation Biologics Mammalian protein expression and characterization tools for next generation biologics next generation for tools characterization and expression protein Mammalian Doctoral Thesis in Biotechnology Mammalian protein expression and characterization tools for next generation biologics NIKLAS THALÉN ISBN TRITACBHFOU: KTH www.kth.se Stockholm, Sweden Mammalian protein expression and characterization tools for next generation biologics NIKLAS THALÉN Academic Dissertation which, with due permission of the KTH Royal Institute of Technology, is submitted for public defence for the Degree of Doctor of Philosophy on June 11th, 2021 at 10:00, F3, Lindstedsvägen 26, våningsplan 2, Sing-Sing, KTH campus, Stockholm. Doctoral Thesis in Biotechnology KTH Royal Institute of Technology Stockholm, Sweden 2021 © Niklas Thalén ISBN 978-91-7873-927-1 TRITA-CBH-FOU-2021:21 Printed by: Universitetsservice US-AB, Sweden 2021 Till Sofia Abstract Protein therapeutics are increasingly important for modern medicine. Novel recombinant proteins developed today can bind towards their target with high specificity and with low adverse effect. This has enabled the treatment of diseases that for a few years ago were deemed uncurable. Discovery of therapeutic proteins is driven through protein engineering, a field that is in constant expansion. And, through artificial construction of recombinant proteins, a large array of diseases can be defeated. The function and quality of these protein therapeutics rely on the correct folding, assembly and residue modification that occurs during their production within a living production cell host. Furthermore, producing them in large quantities are essential for accessibility of the best biopharmaceuticals available. Commonly, mammalian cells are the production host of choice when it comes to production of biopharmaceuticals. Mainly, due to the conserved nature of protein expression pathways within its biological class. Although an ever- growing number of biopharmaceuticals are produced in mammalian cells, there is always room for improvement. Development of novel recombinant protein therapeutics rely on accurate production of the protein. And if this is not achieved, a potential biopharmaceutical will never see the light of day. Furthermore, limited production capabilities can hamper product quality, with less efficacy and increased side-effects as a result. This thesis examines several different pathways for improvements on recombinant protein production for pharmaceutical purposes in mammalian cells. First, the basics of recombinant protein technology and mammalian cell function is outlined. Followed by a summary of six scientific articles revolving within expression and characterization tools for mammalian produced proteins. In paper I, utilization of transcriptomics identifies genes involved in protein expression, which enable the production of a difficult-to-express protein with up to a 150-fold greater activity. Furthermore, in paperIV, transcriptomics reveals genomic differences in a novel cell line that exhibit several fold protein expression capabilities. Besides omics technologies, methods for recombinant protein expression and modification are presented that generate more useable product for several different protein families. And, a protocol for the generation of a pre matured split-GFP variant is presented. Lastly, in paper VI, a mammalian cell display method with an optimized setting that enables precise epitope mapping of glycosylated antigens in a high throughput manner is outlined. With this method, the epitope of four neutralizing antibodies against SARS-CoV-2 is determined. For all of the papers involved within the presented thesis, mammalian cell production of recombinant proteins is the common denominator. Exploring the capabilities of mammalian cell production of current and next-generation biopharmaceuticals is of utter importance to continue the struggle against the gruesome nature of human diseases. Keywords: CHO, Cell line engineering, Protein engineering, GFP, Cell display iv Sammanfattning Proteinläkemedel får stadigt en starkare ställning inom den moderna medicinen. Nya rekombinanta proteiner som utvecklas idag kan binda mot sitt mål med hög specificitet och med få sidoeffekter. Detta har möjliggjort behandling av sjukdomar som för bara några år sen var letala. Utvecklandet av terapeutiska proteiner möjliggörs av proteinteknik, ett relativt ungt område som är i konstant utveckling. Där artificiell konstruktion av rekombinanta proteiner möjliggör bekämpandet av en uppsjö av sjukdomar. För att uppnå rätt funktion och kvalité, så behöver terapeutiska proteiner vara korrekt producerade. Detta sker inom en levande produktions cell, där rätt veckning samt modifikation möjliggör dess konstruktion. Utöver detta så behöver även enorma kvantiteter av biofarmaceutiska läkemedel kunna produceras, för att säkerställa tillgången av de bästa läkemedel som finns att erbjuda. För detta ändamål används främst mammalieceller som produktionsvärd då tillvägagångsättet för proteinkonstruktion är konserverat inom den biologiska klassen. Men även om mammalieceller är bäst lämpade för ändamålet, så finns det ett stort utrymme för förbättringar hos dessa. Utvecklingen annya rekombinanta terapeutiska proteiner är beroende av att tillverkningsprocessen fungerar, och om det ej uppnås så kommer potentiella nya läkemedel aldrig realiseras. Även funktionella tillverkningsprocesser med inneboende begränsningar kan påverka det producerade proteinet negativt, med en lägre och ogynnsam effektivitet som följd. I denna avhandling så undersöks flera olika tillvägagångsätt för att förbättra produktion av rekombinanta proteiner i mammalieceller. Inledningsvis så presenteras det fundamentala inom rekombinant proteinteknik samt mammaliecellers funktion för tillverkande av dessa. Följt av summeringen av sex vetenskapliga artiklar som behandlar metoder för uttryckandet samt karakteriseringen av proteiner tillverkade inom mammalieceller. I artikel I används transkriptomik för identifikation av gener som är involverade i tillverkningen av ett svåruttryckt protein, detta möjliggjorde en 150-faldig ökning av aktivitet hos produkten. Även i artikel IV så identifierades genomiska skillnader kopplade till produktionsökningen hos en ny cellinje med hjälp av transkriptomik. Förutom omik tekniker så presenteras metoder för uttryckandet samt modifieringen av rekombinanta proteiner som genererar mer funktionell produkt för flera olika proteinfamiljer. Även ett protokoll för genererandet av en split-GFP variant där ena delen av molekylen har fått forma fluoroforen i ett tidigare skede presenteras i artikel V. Avslutande så introduceras en optimerad process där ett membran-förankrat antigen möjliggör en detaljrik epitope mappning via mammalieceller. Med denna metod så identifieras inbindningen av fyra antikroppar mot SARS-CoV-2. För samtliga artiklar som presenteras i denna avhandling så är produktion av proteiner inom mammalieceller den gemensamma nämnaren. Utforskandet av möjligheterna inom produktion av rekombinanta proteiner i mammalieceller är av yttersta vikt för att producera funktionella biofarmaceutiska läkemedel både idag samt i framtiden. Vilket möjliggör vidare framgångar i förhindrandet av sjukdomars lidande. v Popular science summary Proteins are one of the fundamental building blocks of life. And our own existence was only made possible, with their emergence some 4 billion years ago. This unique macromolecule exists in a huge variety within all living things. They enable our eyes to process light into vison, carries oxygen to our muscles, and prevents infections when we get ill. With something of this importance, it is easy to see that if errors occur, catastrophic effects might follow. And sadly, errors do occur, and this is often the case for various diseases, ranging from cancer to Alzheimer’s and diabetes. Actually, most known diseases are linked to protein malfunction. However, over the last three decades, scientific advancement within the field of biotechnology have enabled artificial proteins to be a novel cure of many of these diseases. These protein drugs are often called biopharmaceuticals or biologics. And, unlike classical chemical drugs, biologics are produced in living cells. And with the emergence of biologics, vast advancements have been made for modern medicine. Diseases that were fatal just a few years ago, are now treatable. The first biopharmaceutical produced was insulin, a protein that is lacking in patients that suffer from diabetes. This was a huge breakthrough for modern medicine and following this, more and more advanced biopharmaceuticals have been developed, and the pace of innovation is likely not going to decrease in speed. For the first production of insulin, a bacterium was used as the living cell that produced the drug. And although bacterium is an excellent production cell, when more complex biological drugs were developed, they were just not up to the task. This comes from the evolutionary differences between living organisms on earth. Bacterium and humans are simply not that alike. However, mammalian cells are. And rats, hamsters, or human cells cultivated within a lab, are in very close resemblance to all humans on the planet. Therefore, mammalian cells have gained a special position for the production of biologics. And they are routinely used for the assembly of the most complex drugs targeting cancer, rheumatoid arthritis, or multiple sclerosis. Although mammalian cells are capable of
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