| 1. |
- Aniander, Gustav, et al.
(författare)
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Enhanced titer and quality of IgGs through alterations at the translation initiation sequence
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Enhancing recombinant expression in mammalian cells remains an important and valuable goal. Much work has been done to develop cell lines, growth conditions, and expression vectors. However, some parts of the expression vector have been comparatively neglected in this quest for improvement. One such part is the translation initiation site (TIS) sequence, which drives translation initiation by increasing ribosomal recognition of the start codon. Using earlier work on what nucleotides could be exchanged for increased expression, we here present a novel TIS sequence. This TIS sequence increased titer in transient settings 4-fold while yielding 13-33 % increase in titer in final selected stable clones. This increase also comes with an increase in quality through reduction in non-paired chains, shift in charged species distribution, whilst maintaining the glycosylation profile. This increase in both titer and quality in transient and stable settings showing that TIS sequence engineering is of great interest for optimizing recombinant production in mammalian cells.
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| 2. |
- Aniander, Gustav
(författare)
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Improved candidate screening through tailored co-culture assays and precise tuning of protein expression
- 2024
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The field of biopharmaceuticals is a rapidly growing one. In the last ten years the number of approved biopharmaceuticals has more than doubled. A major hurdle to overcome for increased availability of all the new, effective biopharmaceuticals is the cost of treatment. Much of this can be attributed to the sheer time required for their development. Owing to this, interest in improvements to the biopharmaceuticals and their development process has also rapidly increased. As costs increase the further into development a drug candidate progresses, increasing the fidelity of screening at early stages could alleviate some of the exorbitant costs of development.In paper I, we showcase a novel way of targeting the tumor microenvironment (TME) to allow for TMElocalized CD40 activation. This is of interest as CD40 agonists have shown great potential for immune activation, but with systemic activation leading to severe adverse effects. The localized activation is achieved through the construction of an affinity fusion protein termed an AffiMab through fusion of a platelet derived growth factor receptor beta (PDGFRβ) targeting affibody to the heavy chain of a CD40 agonistic monoclonal antibody (mAb). We demonstrate PDGFRβ-dependent activation in a variety of assays, showing that the approach merits further investigation.Building on the activation assays set up in paper I, we aim to generate an in vitro screening platform for immune cell engagers in paper II. Screening candidates for on-target off-tumor activation is essential, as such activation would lead to adverse effects and be a doselimiting factor. To screen for this, we construct a series of plasmids which upon transfecting cells allow for different levels of a cell-surface target protein to be expressed, a so-called target density panel. This is achieved through the use of hairpin forming elements in the 5’ untranslated region of the mRNA dubbed regulatory elements (RgEs). Through use of different RgEs, we show that a target density panel can be generated and validate it in activation assays with the AffiMab developed in paper I. The platforms’ uniform cell surface background due to all different levels of target being expressed in the same host cell line and tunability through use of different RgEs are features that make it interesting for further research.Finally in paper III, we construct and test an improved translation initiation site (TIS) sequence. Using previous studies on the impact of the nucleotides in the sequence on the efficacy of the TIS, we constructed a novel sequence, TISNOV. This sequence enhanced titer and quality for recombinant production of IgG1 and IgG4 in both stable and transient settings. Further research into other TIS sequences and their uses in regulating protein expression, as well as usage of the TISNOV to improve expression of difficult to express proteins such as bispecifics remain interesting.In conclusion this thesis focuses on different manners to improve and hasten development of new biopharmaceuticals through usage of new workflows, platforms, and genetic engineering strategies.
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| 3. |
- Eisenhut, P., et al.
(författare)
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Systematic use of synthetic 5'-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories
- 2020
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 48:20
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Tidskriftsartikel (refereegranskat)abstract
- Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed 'regulation elements (RgE)', in the 5'-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5'-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5'-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.
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| 4. |
- Hendrikse, Natalie, et al.
(författare)
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Ancestral lysosomal enzymes with increased activity harbor therapeutic potential for treatment of Hunter syndrome
- 2021
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Ingår i: ISCIENCE. - : Elsevier BV. - 2589-0042. ; 24:3
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Tidskriftsartikel (refereegranskat)abstract
- We show the successful application of ancestral sequence reconstruction to enhance the activity of iduronate-2-sulfatase (IDS), thereby increasing its therapeutic potential for the treatment of Hunter syndrome-a lysosomal storage disease caused by impaired function of IDS. Current treatment, enzyme replacement therapy with recombinant human IDS, does not alleviate all symptoms, and an unmet medical need remains. We reconstructed putative ancestral sequences of mammalian IDS and compared them with extant IDS. Some ancestral variants displayed up to 2-fold higher activity than human IDS in in vitro assays and cleared more substrate in ex vivo experiments in patient fibroblasts. This could potentially allow for lower dosage or enhanced therapeutic effect in enzyme replacement therapy, thereby improving treatment outcomes and cost efficiency, as well as reducing treatment burden. In summary, we showed that ancestral sequence reconstruction can be applied to lysosomal enzymes that function in concert with modern enzymes and receptors in cells.
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| 5. |
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| 6. |
- Lundqvist, Magnus, et al.
(författare)
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Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion
- 2019
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1-10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1-10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1-10 in analyses. The pre-maturated GFP 1-10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1-10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1-10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.
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| 7. |
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| 8. |
- Mebrahtu, Aman, et al.
(författare)
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Co-culture platform for tuning of cancer receptor density allows for evaluation of bispecific immune cell engagers
- 2024
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 79, s. 120-126
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Tidskriftsartikel (refereegranskat)abstract
- Cancer immunotherapy, where a patient's immune system is harnessed to eradicate cancer cells selectively, is a leading strategy for cancer treatment. However, successes with immune checkpoint inhibitors (ICI) are hampered by reported systemic and organ-specific toxicities and by two-thirds of the patients being non-responders or subsequently acquiring resistance to approved ICIs. Hence substantial efforts are invested in discovering novel targeted immunotherapies aimed at reduced side-effects and improved potency. One way is utilizing the dual targeting feature of bispecific antibodies, which have made them increasingly popular for cancer immunotherapy. Easy and predictive screening methods for activation ranking of candidate drugs in tumor contra non-tumor environments are however lacking. Herein, we present a cell-based assay mimicking the tumor microenvironment by co-culturing B cells with engineered human embryonic kidney 293 T cells (HEK293T), presenting a controllable density of platelet-derived growth factor receptor β (PDGFRβ). A target density panel with three different surface protein levels on HEK293T cells was established by genetic constructs carrying regulatory elements limiting RNA translation of PDGFRβ. We employed a bispecific antibody-affibody construct called an AffiMab capable of binding PDGFRβ on cancer cells and CD40 expressed by B cells as a model. Specific activation of CD40-mediated signaling of immune cells was demonstrated with the two highest receptor-expressing cell lines, Level 2/3 and Level 4, while low-to-none in the low-expressing cell lines. The concept of receptor tuning and the presented co-culture protocol may be of general utility for assessing and developing novel bi-specific antibodies for immuno-oncology applications.
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| 9. |
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| 10. |
- Moradi, Mona, et al.
(författare)
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Autophagy and intracellular product degradation genes identified by systems biology analysis reduce aggregation of bispecific antibody in CHO cells
- 2022
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 68, s. 68-76
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Tidskriftsartikel (refereegranskat)abstract
- Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of these products. Pairs of stable Chinese hamster ovary (CHO) cell lines produced two difficult-to-express bispecific antibodies with different levels of aggregated product (10-75% aggregate) in a miniaturised bioreactor system. Here, transcriptome analysis was used to interpret the biological causes for the aggregation and to identify strategies to improve product yield and quality. Differential expression-and gene set analysis revealed upregulated proteasomal degradation, unfolded protein response and autophagy processes to be correlated with reduced protein aggregation. Fourteen candidate genes with the potential to reduce aggregation were co expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AKT1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased titres of the anti-HER2 monoclonal antibody trastuzumab by 50% during transient expression. It is suggested that this approach could be of general use for defining aggregation bottlenecks in CHO cells.
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| 11. |
- Moradi, Mona, et al.
(författare)
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Autophagy and intracellular product degradation genes reduce aggregation of bispecific antibody in CHO cells with a high translational burden
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of the product. Pairs of stable Chinese hamster ovary cell lines produced two difficult- to-express bispecific antibodies with different levels of aggregated product (10-75% aggregate) in a miniaturized bioreactor system. Here, we analyse the cellular response and link to product aggregation by comparative transcriptome analysis of these CHO cells, to define biological causes and infer strategies to improve yield and quality. Differential expression- and gene set analysis revealed upregulated proteosomal degradation, unfolded protein response and autophagy processes to be correlated with reduction of protein aggregation. Fourteen candidate genes with potential to reduce aggregation were co-expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AK1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased trastuzumab titres by 50% each during transient expression. We suggest our approach to be of general use for defining aggregation bottlenecks in CHO.
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| 12. |
- Thalén, Niklas, et al.
(författare)
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Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping
- 2024
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Ingår i: Communications Biology. - : Springer Nature. - 2399-3642. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.
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| 13. |
- Thalén, Niklas, et al.
(författare)
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Mammalian cell surface display system for conformational epitope determination of SARS-CoV-2 neutralizing antibodies
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Cell surface display of recombinant proteins for epitope mapping has become a powerful tool for biotechnology and biomedical applications. One critical aspect for identifying the full and correct epitope is to present the correctly folded and post-translationally modified protein antigen on the cell surface for true epitope-paratope interaction. Being the gold standard for production of complex proteins, Chinese hamster ovarian cells are here used for a mammalian cell display system. Additionally, to enable a fast workflow for alanine substitution of a large panel of clones, a web-based design tool and automated cloning process was established. A proof-of-concept is presented with the aligned epitope of the crystal structure for the CR3022 antibody with our mammalian cell display determined epitope. Furthermore, three additional antibody epitopes against SARS-CoV-2 RBD are presented. This study presents a detailed view of epitope-paratope interactions, which is of great importance for future clinical interventions against SARS-CoV-2.
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| 14. |
- Thalén, Niklas, 1985-
(författare)
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Mammalian protein expression and characterization tools for next generation biologics
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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 evergrowing 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 paper IV, 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.
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| 15. |
- Thalén, Niklas, 1985-, et al.
(författare)
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Systems biology greatly improve activity of secreted therapeutic sulfatase in CHO bioprocess
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. However, such recombinant production of sulfatases suffers greatly from low product activity and yield, further limiting accessibility for patient groups. Here, we have addressed this problem by defining key-proteins necessary for active sulfatase secretion by comparison of CHO clones with different levels of production of active sulfatase. Quantitative transcriptomic analysis highlighted 14 key genes associated with sulfatase production, and experimental validation by co-expression improved the sulfatase enzyme activity by up to 150-fold. Furthermore, a correlation between product mRNA levels and sulfatase activity were observed and expression with lower activity promoters showed an increased in sulfatase activity. The workflow devised is general and we propose it to be useful for resolving bottlenecks in cellular machineries for improvement of cell factories for other biologics as well.
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| 16. |
- Thalén, Niklas, et al.
(författare)
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Tuning of CHO secretional machinery improve activity of secreted therapeutic sulfatase 150-fold
- 2024
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 81, s. 157-166
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Tidskriftsartikel (refereegranskat)abstract
- Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. The recombinant production of such sulfatases suffers greatly from both low product activity and yield, further limiting accessibility for patient groups. To mitigate the low product activity, we have investigated cellular properties through computational evaluation of cultures with varying media conditions and comparison of two CHO clones with different levels of one active sulfatase variant. Transcriptome analysis identified 18 genes in secretory pathways correlating with increased sulfatase production. Experimental validation by upregulation of a set of three key genes improved the specific enzymatic activity at varying degree up to 150-fold in another sulfatase variant, broadcasting general production benefits. We also identified a correlation between product mRNA levels and sulfatase activity that generated an increase in sulfatase activity when expressed with a weaker promoter. Furthermore, we suggest that our proposed workflow for resolving bottlenecks in cellular machineries, to be useful for improvements of cell factories for other biologics as well.
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| 17. |
- Thalén, Niklas, et al.
(författare)
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ULK1 knockout cell line downregulates autophagy, upregulates recombinant transcript and improves protein secretion
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- To meet the ever-growing demand for effective, safe, and affordable protein therapeutics, decades of bioprocessing innovations and cell engineering modifications have vastly improved the production of recombinant proteins. Recently, new genomic technologies allow more targeted approaches in cell line development where most effort have been aimed at reducing toxic byproducts or regulate specific traits, such as apoptosis or glycosylation. By targeting a general metabolic or processing pathway a broader protein expression increase could be achieved. In order to identify a more general cell engineered platform, a small molecule screen for enhanced protein expression of three model proteins was performed. From this, ULK1, the key initiator of autophagy, emerged as an important component for improved recombinant expression. Autophagy is a large process within the cell that restores cell homeostasis during cell stress, and knockout of ULK1 improved protein expression 3-fold in a stable Cripto-Fc producing cell. Transcriptomic analysis showed a downregulation of autophagy and upregulation of transcriptional processes when ULK1 was removed. Processes within the host cell that are of a general cell maintenance character have great potential to be engineered into a universal manufacturing platform, here shown through the prevention of autophagy.
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