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Publications (10 of 31) Show all publications
Thalén, N., Karlander, M., Lundqvist, M., Persson, H., Hofström, C., Turunen, S. P., . . . Rockberg, J. (2024). Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping. Communications Biology, 7(1), Article ID 805.
Open this publication in new window or tab >>Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping
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2024 (English)In: Communications Biology, E-ISSN 2399-3642, Vol. 7, no 1, article id 805Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Springer Nature, 2024
National Category
Biochemistry Molecular Biology
Identifiers
urn:nbn:se:kth:diva-350701 (URN)10.1038/s42003-024-06508-8 (DOI)001262592300006 ()38961245 (PubMedID)2-s2.0-85197485847 (Scopus ID)
Note

QC 20240719

Available from: 2024-07-17 Created: 2024-07-17 Last updated: 2025-02-20Bibliographically approved
Thalén, N., Moradi, M., Lundqvist, M., Rodhe, J., Andersson, M., Bidkhori, G., . . . Rockberg, J. (2024). Tuning of CHO secretional machinery improve activity of secreted therapeutic sulfatase 150-fold. Metabolic engineering, 81, 157-166
Open this publication in new window or tab >>Tuning of CHO secretional machinery improve activity of secreted therapeutic sulfatase 150-fold
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2024 (English)In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 81, p. 157-166Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier BV, 2024
Keywords
CHO, Sulfatase, Systems biology, Transcriptomics
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-341758 (URN)10.1016/j.ymben.2023.12.003 (DOI)001138624600001 ()38081506 (PubMedID)2-s2.0-85179839715 (Scopus ID)
Note

QC 20240102

Available from: 2024-01-02 Created: 2024-01-02 Last updated: 2024-01-22Bibliographically approved
Leitao, C. D., Mestre Borras, A., Jonsson, A., Malm, M., Kronqvist, N., Fleetwood, F., . . . Lindberg, H. (2023). Display of a naïve affibody library on staphylococci for selection of binders by means of flow cytometry sorting. Biochemical and Biophysical Research Communications - BBRC, 655, 75-81
Open this publication in new window or tab >>Display of a naïve affibody library on staphylococci for selection of binders by means of flow cytometry sorting
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2023 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 655, p. 75-81Article in journal (Refereed) Published
Abstract [en]

Within the field of combinatorial protein engineering there is a great demand for robust high-throughput selection platforms that allow for unbiased protein library display, affinity-based screening, and amplification of selected clones. We have previously described the development of a staphylococcal display system used for displaying both alternative-scaffolds and antibody-derived pro-teins. In this study, the objective was to generate an improved expression vector for displaying and screening a high-complexity naive affibody library, and to facilitate downstream validation of isolated clones. A high-affinity normalization tag, consisting of two ABD-moieties, was introduced to simplify off-rate screening procedures. In addition, the vector was furnished with a TEV protease substrate recog-nition sequence upstream of the protein library which enables proteolytic processing of the displayed construct for improved binding signal. In the library design, 13 of the 58 surface-exposed amino acid positions were selected for full randomization (except proline and cysteine) using trinucleotide tech-nology. The genetic library was successfully transformed to Staphylococcus carnosus cells, generating a protein library exceeding 109 members. De novo selections against three target proteins (CD14, MAPK9 and the affibody ZEGFR:2377) were successfully performed using magnetic bead-based capture followed by flow-cytometric sorting, yielding affibody molecules binding their respective target with nanomolar affinity. Taken together, the results demonstrate the feasibility of the staphylococcal display system and the proposed selection procedure to generate new affibody molecules with high affinity.

Place, publisher, year, edition, pages
Elsevier BV, 2023
Keywords
Affibody molecules, Bacterial surface display, Combinatorial protein engineering, Flow cytometry, Staphyloccoccus carnosus
National Category
Biochemistry Molecular Biology
Identifiers
urn:nbn:se:kth:diva-327166 (URN)10.1016/j.bbrc.2023.03.003 (DOI)000972618100001 ()36933310 (PubMedID)2-s2.0-85150381236 (Scopus ID)
Note

QC 20230523

Available from: 2023-05-23 Created: 2023-05-23 Last updated: 2025-02-20Bibliographically approved
Moradi, M., Lundqvist, M., Harris, C., Malm, M., Volk, A.-L., Thalén, N., . . . Rockberg, J. (2022). Autophagy and intracellular product degradation genes identified by systems biology analysis reduce aggregation of bispecific antibody in CHO cells. New Biotechnology, 68, 68-76
Open this publication in new window or tab >>Autophagy and intracellular product degradation genes identified by systems biology analysis reduce aggregation of bispecific antibody in CHO cells
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2022 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 68, p. 68-76Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier BV, 2022
Keywords
CHO cells, Aggregation, Autophagy, ER stress, Bispecific antibody, System biology
National Category
Cell and Molecular Biology Biochemistry Molecular Biology
Identifiers
urn:nbn:se:kth:diva-310578 (URN)10.1016/j.nbt.2022.01.010 (DOI)000766630900008 ()35123066 (PubMedID)2-s2.0-85124238085 (Scopus ID)
Note

QC 20220406

Available from: 2022-04-06 Created: 2022-04-06 Last updated: 2025-02-20Bibliographically approved
Malm, M., Kuo, C.-C., Moradi, M., Mebrahtu, A., Wistbacka, N., Razavi, R., . . . Rockberg, J. (2022). Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins. Metabolic engineering, 72, 171-187
Open this publication in new window or tab >>Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins
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2022 (English)In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 72, p. 171-187Article in journal (Refereed) Published
Abstract [en]

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.

Place, publisher, year, edition, pages
Elsevier BV, 2022
Keywords
HEK293, CHO, Bioproduction, Protein secretion, Transcriptomics, Differential gene expression analysis, Secretory pathway
National Category
Genetics and Genomics
Identifiers
urn:nbn:se:kth:diva-313037 (URN)10.1016/j.ymben.2022.03.009 (DOI)000793760100001 ()35301123 (PubMedID)2-s2.0-85126895070 (Scopus ID)
Note

QC 20220531

Available from: 2022-05-31 Created: 2022-05-31 Last updated: 2025-02-07Bibliographically approved
Saghaleyni, R., Malm, M., Moruzzi, N., Zrimec, J., Razavi, R., Wistbacka, N., . . . Rockberg, J. (2022). Report Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells. Cell Reports, 39(11), 110936, Article ID 110936.
Open this publication in new window or tab >>Report Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells
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2022 (English)In: Cell Reports, E-ISSN 2211-1247, Vol. 39, no 11, p. 110936-, article id 110936Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier BV, 2022
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-315704 (URN)10.1016/j.celrep.2022.110936 (DOI)000817793600006 ()35705050 (PubMedID)2-s2.0-85131966582 (Scopus ID)
Note

QC 20220718

Available from: 2022-07-18 Created: 2022-07-18 Last updated: 2024-01-17Bibliographically approved
Malm, M., Volk, A.-L., Karlander, M., Lundqvist, M., Wistbacka, N., Razavi, R., . . . Rockberg, J. (2021). Improving targeting and yield of AAV by capsid and cell engineering. Human Gene Therapy, 32(19-20), A119-A120
Open this publication in new window or tab >>Improving targeting and yield of AAV by capsid and cell engineering
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2021 (English)In: Human Gene Therapy, ISSN 1043-0342, E-ISSN 1557-7422, Vol. 32, no 19-20, p. A119-A120Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
MARY ANN LIEBERT, INC, 2021
National Category
Genetics and Genomics Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-315551 (URN)000811305600350 ()
Note

QC 20220708

Available from: 2022-07-08 Created: 2022-07-08 Last updated: 2025-02-01Bibliographically approved
Malm, M., Saghaleyni, R., Lundqvist, M., Giudici, M., Chotteau, V., Field, R., . . . Rockberg, J. (2020). Evolution from adherent to suspension: systems biology of HEK293 cell line development. Scientific Reports, 10(1), Article ID 18996.
Open this publication in new window or tab >>Evolution from adherent to suspension: systems biology of HEK293 cell line development
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2020 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 10, no 1, article id 18996Article in journal (Refereed) Published
Abstract [en]

The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

Place, publisher, year, edition, pages
Springer Nature, 2020
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-287517 (URN)10.1038/s41598-020-76137-8 (DOI)000587637100003 ()33149219 (PubMedID)2-s2.0-85090610994 (Scopus ID)
Note

QC 20210303

Correction: 10.1038/s41598-021-85105-9 (Scopus: 2-s2.0-85101943219)

Available from: 2021-03-03 Created: 2021-03-03 Last updated: 2022-09-15Bibliographically approved
Zhan, C., Bidkhori, G., Schwarz, H., Malm, M., Mebrahtu, A., Field, R., . . . Chotteau, V. (2020). Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells. iScience, 23(11), Article ID 101653.
Open this publication in new window or tab >>Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells
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2020 (English)In: iScience, ISSN 2589-0042, Vol. 23, no 11, article id 101653Article in journal (Refereed) Published
Abstract [en]

Human embryonic kidney cells HEK293 can be used for the production of therapeutic glycoproteins requiring human post-translational modifications. High cell density perfusion processes are advantageous for such production but are challenging due to the shear sensitivity of HEK293 cells. To understand the impact of hollow filter cell separation devices, cells were cultured in bioreactors operated with tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) at various flow rates. The average theoretical velocity profile in these devices showed a lower shear stress for ATF by a factor 0.637 compared to TFF. This was experimentally validated and, furthermore, transcriptomic evaluation provided insights into the underlying cellular processes. High shear caused cellular stress leading to apoptosis by three pathways, i.e. endoplasmic reticulum stress, cytoskeleton reorganization, and extrinsic signaling pathways. Positive effects of mild shear stress were observed, with increased recombinant erythropoietin production and increased gene expression associated with transcription and protein phosphorylation.

Place, publisher, year, edition, pages
Elsevier BV, 2020
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-289557 (URN)10.1016/j.isci.2020.101653 (DOI)000594402400008 ()33145483 (PubMedID)2-s2.0-85094208423 (Scopus ID)
Note

QC 20210203

Available from: 2021-02-03 Created: 2021-02-03 Last updated: 2024-03-18Bibliographically approved
Schwarz, H., Zhang, Y., Zhan, C., Malm, M., Field, R., Turner, R., . . . Chotteau, V. (2020). Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin. Journal of Biotechnology, 309, 44-52
Open this publication in new window or tab >>Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin
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2020 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 309, p. 44-52Article in journal (Refereed) Published
Abstract [en]

Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 10(8) cells/mL in the recent years. As the majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells, intensified perfusion processes have been mainly developed for this type of host cell line. However, the use of CHO cells can result in non-human posttranslational modifications of the protein of interest, which may be disadvantageous compared with human cell lines. In this study, we developed a high cell density perfusion process of Human Embryonic Kidney (HEK293) cells producing recombinant human Erythropoietin (rhEPO). Firstly, a small-scale perfusion system from commercial bench-top screening bioreactors was developed for < 250 mL working volume. Then, after the first trial runs with CHO cells, the system was modified for HEK293 cells (more sensitive than CHO cells) to achieve a higher oxygen transfer under mild aeration and agitation conditions. Steady states for medium (20 x 10(6) cells/mL) and high cell densities (80 x 10(6) cells/mL), normal process temperature (37 degrees C) and mild hypothermia (33 degrees C) as well as different cell specific perfusion rates (CSPR) from 10 to 60 pL/cell/day were applied to study the performance of the culture. The volumetric productivity was maximized for the high cell density steady state but decreased when an extremely low CSPR of 10 pL/cell/day was applied. The shift from high to low CSPR strongly reduced the nutrient uptake rates. The results from our study show that human cell lines, such as HEK293 can be used for intensified perfusion processes.

Place, publisher, year, edition, pages
Elsevier, 2020
Keywords
Chinese Hamster Ovary (CHO) cells, Erythropoietin, High cell density culture, Human embryonic kidney 239 (HEK293) cells, Perfusion process, Small-scale bioreactor
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-268782 (URN)10.1016/j.jbiotec.2019.12.017 (DOI)000510824700005 ()31891733 (PubMedID)2-s2.0-85077033718 (Scopus ID)
Note

QC 20200226

Available from: 2020-02-26 Created: 2020-02-26 Last updated: 2024-03-18Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0003-1763-9073

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