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  • 1.
    Andersson, Ken G
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Rosestedt, Maria
    Varasteh, Zohreh
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Sandström, Mattias
    Tolmachev, Vladimir
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Orlova, Anna
    Comparative evaluation of 111In-labeled NOTA‑conjugated affibody molecules for visualization of HER3 expression in malignant tumors2015Inngår i: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 34, nr 2, s. 1042-8Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been associated with resistance to a variety of anticancer therapies. Several anti-HER3 monoclonal antibodies are currently under pre-clinical and clinical development aiming to overcome HER3-mediated resistance. Radionuclide molecular imaging of HER3 expression may improve treatment by allowing the selection of suitable patients for HER3-targeted therapy. Affibody molecules are a class of small (7kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. In a recent study, we selected affibody molecules with affinity to HER3 at a low picomolar range. The aim of the present study was to develop an anti-HER3 affibody molecule suitable for labeling with radiometals. The HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA HER3-specific affibody molecules were labeled with indium‑111 (111In) and assessed invitro and invivo for imaging properties using single photon emission computed tomography (SPECT). Labeling of HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA with 111In provided stable conjugates. Invitro cell tests demonstrated specific binding of the two conjugates to HER3-expressing BT‑474 breast carcinoma cells. In mice bearing BT‑474 xenografts, the tumor uptake of the two conjugates was receptor‑specific. Direct invivo comparison of 111In-HEHEHE-Z08698-NOTA and 111In-HEHEHE-Z08699‑NOTA demonstrated that the two conjugates provided equal radioactivity uptake in tumors, although the tumor-to-blood ratio was improved for 111In-HEHEHE-Z08698-NOTA [12±3 vs. 8±1, 4h post injection (p.i.)] due to more efficient blood clearance. 111In-HEHEHE-Z08698-NOTA is a promising candidate for imaging of HER3-expression in malignant tumors using SPECT. Results of the present study indicate that this conjugate could be used for patient stratification for anti-HER3 therapy.

  • 2.
    Göstring, Lovisa
    et al.
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Stockholm, Sweden.
    Frejd, Fredrik Y.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Gedda, Lars
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala Sweden.
    Cellular Effects of HER3-Specific Affibody Molecules2012Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 6, s. e40023-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Recent studies have led to the recognition of the epidermal growth factor receptor HER3 as a key player in cancer, and consequently this receptor has gained increased interest as a target for cancer therapy. We have previously generated several Affibody molecules with subnanomolar affinity for the HER3 receptor. Here, we investigate the effects of two of these HER3-specific Affibody molecules, Z05416 and Z05417, on different HER3-overexpressing cancer cell lines. Using flow cytometry and confocal microscopy, the Affibody molecules were shown to bind to HER3 on three different cell lines. Furthermore, the receptor binding of the natural ligand heregulin (HRG) was blocked by addition of Affibody molecules. In addition, both molecules suppressed HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, as well as HER3 phosphorylation in constantly HER2-activated SKBR-3 cells. Importantly, Western blot analysis also revealed that HRG-induced downstream signalling through the Ras-MAPK pathway as well as the PI3K-Akt pathway was blocked by the Affibody molecules. Finally, in an in vitro proliferation assay, the two Affibody molecules demonstrated complete inhibition of HRG-induced cancer cell growth. Taken together, our findings demonstrate that Z05416 and Z05417 exert an anti-proliferative effect on two breast cancer cell lines by inhibiting HRG-induced phosphorylation of HER3, suggesting that the Affibody molecules are promising candidates for future HER3-targeted cancer therapy.

  • 3.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Göstring, Lovisa
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Gunneriusson, Elin
    Nilsson, Martin
    Affibody AB, Stockholm, Sweden.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Stockholm, Sweden.
    Gedda, Lars
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala Sweden.
    Frejd, Fredrik Y.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules2011Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 24, nr 4, s. 385-396Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.

  • 4.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Staphylococcal surface display in combinatorial protein engineering and epitope mapping of antibodies2010Inngår i: Recent Patents on Biotechnology, ISSN 1872-2083, Vol. 4, nr 3, s. 171-182Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The field of combinatorial protein engineering for generation of new affinity proteins started in the mid 80s by the development of phage display. Although phage display is a prime example of a simple yet highly efficient method, manifested by still being the standard technique 25 years later, new alternative technologies are available today. One of the more successful new display technologies is cell display. Here we review the field of cell display for directed evolution purposes, with focus on a recently developed method employing Gram-positive staphylococci as display host. Patents on the most commonly used cell display systems and on different modifications as well as specific applications of these systems are also included. General strategies for selection of new affinity proteins from cell-displayed libraries are discussed, with detailed examples mainly from studies on the staphylococcal display system. In addition, strategies for characterization of recombinant proteins on the staphylococcal cell surface, with an emphasis on an approach for epitope mapping of antibodies, are included.

  • 5.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Generation and characterization of Affibody molecules targeting HER32013Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In the field of oncology, the ability to target specific tumor cells using highly selective targeting molecules is an attractive and emerging concept. In this context, the epidermal growth factor receptor HER3 has proven central to the biology behind many different human cancers and inhibition of the signaling mediated by this receptor could provide antitumoral effects. Consequently, this receptor has emerged as a suitable target for imaging, functional blocking or delivery of toxic payloads. A promising targeting-molecule for such applications is the small non-immunoglobulin derived Affibody molecule. The work upon which this thesis is based, revolves around HER3 with the aim to generate and characterize Affibody molecules targeting this receptor.

     

    In the first study, HER3-specific Affibody molecules were generated by combinatorial protein engineering using a combined approach where first generation binders were isolated from a phage-displayed naive library, followed by affinity maturation of these binders using a focused staphylococcal surface-displayed library and flow-cytometric cell sorting. This engineering strategy enabled the successful isolation of HER3-specific Affibody molecules with subnanomolar affinities for the receptor and the ability to compete with the natural ligand heregulin (HRG) for binding to HER3. In the second study, the cellular effects of these Affibody molecules were characterization in vitro. The results demonstrated that the ability to inhibit HRG-binding to the receptor translated into inhibition of ligand-induced phosphorylation of HER3, HER2 as well as the downstream signaling molecules Akt and Erk. As a result, the HER3-specific Affibody molecules also inhibited HRG-induced cell growth of two different breast cancer cell lines in vitro. These promising results, suggested that the HER3-targeting Affibody molecules could have a therapeutic effect in tumors that are dependent on ligand-induced signaling of HER3. However, due to the relatively low expression level of HER3 on tumor cells, we explored two different engineering approaches of the HER3-specific Affibody molecules in order to potentially improve its tumor targeting ability. One approach was to construct bispecific Affibody molecules where a HER3- and a HER2-specific Affibody molecule were fused on each side of an albumin-binding domain (ABD). In the third study, one such bispecific construct was shown to have increased ability to inhibit ligand-induced phosphorylation of HER2 and retained ability to inhibit HRG-induced activation of HER3, as compared to the monomeric anti-HER3 Affibody. Another strategy was to further increase the affinity of the HER3-specific Affibody molecules towards the receptor through a semi-rational affinity maturation approach. In the fourth study, a staphylococcal displayed affinity maturation library was screened by FACS using an off-rate selection procedure. This approach resulted in the successful isolation of picomolar HER3-binders with improved potency of inhibiting HRG-induced cell growth as compared to a first generation binder. Moreover, in the fifth study, in vivo characterization of these HER3-specific Affibody molecules was performed in both normal and xenograft mice. The results suggested specific targeting of HER3 in vivo and provided the first evidence of successful tumor imaging using a HER3-specific Affibody. Taken together, the work included in this thesis describes (to our knowledge) the first non-immunoglobulin derived affinity protein targeting HER3, with promising features for both therapeutic and imaging applications.

  • 6.
    Malm, Magdalena
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Bass, Tarek
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Gudmundsdotter, Lindvi
    Lord, Martin
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, Fredrik Y.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension2014Inngår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, nr 9, s. 1215-1222Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Emerging strategies in cancer biotherapy include the generation and application of bispecific antibodies, targeting two tumor-associated antigens for improved tumor selectivity and potency. Here, an alternative format for bispecific molecules was designed and investigated, in which two Affibody molecules were linked by an albumin-binding domain (ABD). Affibody molecules are small (6 kDa) affinity proteins and this new format allows for engineering of molecules with similar function as full-length bispecific antibodies, but in a dramatically smaller size (around eight-fold smaller). The ABD was intended to function both as a tag for affinity purification as well as for in vivo half-life extension in future preclinical and clinical investigations. Affinity-purified bispecific Affibody molecules, targeting HER2 and HER3, showed simultaneous binding to the three target proteins (HER2, HER3, and albumin) when investigated in biosensor assays. Moreover, simultaneous interactions with the receptors and albumin were demonstrated using flow cytometry on cancer cells. The bispecific Affibody molecules were also able to block ligand-induced phosphorylation of the HER receptors, indicating an anti-proliferative effect. We believe that this compact and flexible format has great potential for developing new potent bispecific affinity proteins in the future, as it combines the benefits of a small size (e.g. improved tissue penetration and reduced cost of goods) with a long circulatory half-life.

  • 7.
    Malm, Magdalena
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, F. Y.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Targeting HER3 using mono- and bispecific antibodies or alternative scaffolds2016Inngår i: mAbs, ISSN 1942-0862, E-ISSN 1942-0870, Vol. 8, nr 7, s. 1195-1209Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The human epidermal growth factor receptor 3 (HER3) has in recent years been recognized as a key node in the complex signaling network of many different cancers. It is implicated in de novo and acquired resistance against therapies targeting other growth factor receptors, e.g., EGFR, HER2, and it is a major activator of the PI3K/Akt signaling pathway. Consequently, HER3 has attracted substantial attention, and is today a key target for drugs in clinical development. Sophisticated protein engineering approaches have enabled the generation of a range of different affinity proteins targeting this receptor, including antibodies and alternative scaffolds that are either mono- or bispecific. Here, we describe HER3 and its role as a key tumor target, and give a comprehensive review of HER3-targeted proteins currently in development, including discussions on the opportunities and challenges of targeting this receptor.

  • 8.
    Malm, Magdalena
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Kronqvist, Nina
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Lindberg, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Gudmundsdotter, Lindvi
    Affibody AB, Stockholm, Sweden.
    Bass, Tarek
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, Fredrik Y.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Stockholm, Sweden.
    Varasteh, Zohreh
    Departmaent of Medical Chemistry, Preclinical PET Platform, Uppasala University, Uppsala, Sweden.
    Orlova, Anna
    Departmaent of Medical Chemistry, Preclinical PET Platform, Uppasala University, Uppsala, Sweden.
    Tolmachev, Vladimir
    Unit of Biomedical Radiation Sciences, Uppsala University, Uppsala, Sweden.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification2013Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 5, s. e62791-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 PM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

  • 9. Orlova, A.
    et al.
    Malm, Malin
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Lindberg, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Varasteh, Z.
    Selvaraju, R. K.
    Kronqvist, Nina
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, V.
    Feasibility of radionuclide imaging of HER3-expressing tumours using technetium-99m labeled affibody molecules2013Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, s. S185-S186Artikkel i tidsskrift (Annet vitenskapelig)
  • 10. Orlova, Anna
    et al.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Lindberg, Hanna
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Varasteh, Zohreh
    Rosestedt, Maria
    Tolmachev, Vadimir
    Kronqvist, Nina
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Feasibility of radionuclide imaging of HER3-expressing tumors using affibody molecules2013Inngår i: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 56, s. S11-S11Artikkel i tidsskrift (Annet vitenskapelig)
  • 11. Orlova, Anna
    et al.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Rosestedt, Maria
    Varasteh, Zohreh
    Andersson, Ken
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Selvaraju, Ram Kumar
    Altai, Mohamed
    Honarvar, Hadis
    Strand, Joanna
    Stahl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, Vladimir
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Imaging of HER3-expressing xenografts in mice using a Tc-99m(CO)(3)-HEHEHE-Z(HER3:08699) affibody molecule2014Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, nr 7, s. 1450-1459Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Human epidermal growth factor receptor type 3 (HER3) is a transmembrane receptor tyrosine kinase belonging to the HER (ErbB) receptor family. Membranous expression of HER3 is associated with trastuzumab resistance in breast cancer and the transition to androgen independence in prostate cancer. Imaging of HER3 expression in malignant tumors may provide important diagnostic information that can influence patient management. Affibody molecules with low picomolar affinity to HER3 were recently selected. The aim of this study was to investigate the feasibility of HER3 imaging using radiolabeled Affibody molecules. A HER3-binding Affibody molecule, Z(08699), with a HEHEHE-tag on N-terminus was labeled with Tc-99m(CO)(3) using an IsoLink kit. In vitro and in vivo binding specificity and the cellular processing of the labeled binder were evaluated. Biodistribution of Tc-99m(CO)(3)-HEHEHE-Z(08699) was studied over time in mice bearing HER3-expressing xenografts. HEHEHE-Z(08699) was labeled with Tc-99m(CO)(3) with an isolated yield of > 80 % and a purity of > 99 %. Binding of Tc-99m(CO)(3)-HEHEHE-Z(08699) was specific to BT474 and MCF7 (breast cancer), and LS174T (colon cancer) cells. Cellular processing showed rapid binding and relatively quick internalization of the receptor/Affibody molecule complex (70 % of cell-associated radioactivity was internalized after 24 h). The tumor targeting was receptor mediated and the excretion was predominantly renal. Receptor-mediated uptake was also found in the liver, lung, stomach, intestine, and salivary glands. At 4 h pi, tumor-to-blood ratios were 7 +/- 3 for BT474, and 6 +/- 2 for LS174T xenografts. LS174T tumors were visualized by microSPECT 4 h pi. The results of this study suggest the feasibility of HER3-imaging in malignant tumors using Affibody molecules.

  • 12.
    Schwarz, Hubert
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Zhang, Ye
    KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Zhan, Caijuan
    KTH, Skolan för teknikvetenskap (SCI). KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Malm, Magdalena
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Field, Raymond
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Turner, Richard
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Sellick, Christopher
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Varley, Paul
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Chotteau, Véronique
    KTH, Skolan för teknikvetenskap (SCI).
    Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant ErythropoietinManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 108 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 106 cells/mL) and high cell densities (80 x 106 cells/mL), normal process temperature (37 °C) and mild hypothermia (33 °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. 

  • 13.
    Zhan, Caijuan
    et al.
    KTH, Skolan för teknikvetenskap (SCI). KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Hubert, Schwarz
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Malm, Magdalena
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Sellick, Christopher
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Chotteau, Véronique
    KTH, Skolan för teknikvetenskap (SCI). KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Hydrodynamic shear stress in hollow filter for perfusion culture of human cellsManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    High cell density perfusion process is an economical way to produce biopharmaceuticals at high yield. To achieve high density of healthy cells, the cell culture conditions should be free from mechanically detriment. Human embryonic kidney (HEK) K293 cells, interesting for the production of therapeutic glycoproteins, are known as shear sensitive. In order to obtain the optimal hydrodynamics conditions with reduced mechanical damage, we investigated the fact of the shear stress compatible with HEK293 cells. We reviewed hollow filter based tangential flow filtration strategies, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). We studied shear stress introduced by these two flow filtration methods. By theoretical study, we obtained that lower shear stress introduced by alternating tangential flow filtration result in lower average shear stress comparing to tangential flow filtration with same flow rate.  In our experimental runs, we achieved different shear stress levels by applying different flow rates. 5-Days batch cultivations were performed to examine the influence of shear stress on cell growing and metabolic behaviour. We identified that the shear stress potentially reduce the growth rate and productivity of HEK293 cells and found the cell metabolism associated with shear stress levels. By documenting these cell responses to shear stress, we confirmed our theoretical results and could further optimize the hydrodynamic conditions for perfusion process of HEK 293 cells. 

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