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      Differences in the production of hyperglycosylated IFN alpha in CHO and HEK 293 cells

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      BMC Proceedings

      BioMed Central

      23rd European Society for Animal Cell Technology (ESACT) Meeting: Better Cells for Better Health

      23-26 June 2013

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          Abstract

          Background IFN alpha is an important cytokine of the immune system. It has the ability to interfere with virus replication exerting antiviral activity. Moreover, it displays antiproliferative activity and can profoundly modulate the immune response. IFN4N (or hyperglycosylated IFN alpha) is an IFN-alpha2b mutein developed in our laboratory using glycoengineering strategies. This molecule contains 4 potential N-glycosylation sites together with the natural O-glycosylation site in Thr106 [1]. The resulting N- and O-glycosylated protein shows higher apparent molecular mass and longer plasmatic half-life compared to the non-glycosylated IFN-alpha produced in bacterial systems and used for clinical applications. As a consequence, the correct glycosylation of our modified cytokine is very important for its in vivo activity. For this reason, it is of great relevance the evaluation of different mammalian host cells for its production. While hamster-derived CHO cells are widely used for large scale production of recombinant therapeutic glycoproteins, human HEK cells are a promising system because they are easy to grow and transfect [2]. In this work, we performed a comparison between both production systems in terms of cell growth, culture parameters and specific productivity of hyperglycosylated IFN alpha. Results Lentiviral vectors containing the sequence of IFN4N were assembled and employed for the transduction of CHO-K1 and HEK 293T cells. The recombinant cell lines were subjected to a process of selective pressure using increasing concentrations of puromycin. The CHO-IFN4N and HEK-IFN4N producing cell lines resistant to the highest concentration of puromycin showed the highest productivity of IFN4N. In particular, the CHO-IFN4N cell line was resistant to 350 μg/ml of puromycin and it showed a specific productivity of 817 ± 134 ng.106cell-1.day-1, which represents an 8-fold increment compared to the parental line. The HEK-IFN4N cell line was resistant to 200 ug/ml of puromycin and showed a 15-fold increment in the specific productivity compared to the parental line, reaching a value of 1,490 ± 332 ng.106cell-1.day-1. In both cases, complete culture death was achieved at higher puromycin concentrations. The specific productivity of IFN4N of HEK 293T cell line duplicated the value obtained for the CHO-K1 cell line, and it was achieved at a lower concentration of puromycin, making the selection process shorter (Figure 1). Figure 1 Comparison between the specific productivity of the CHO-IFN4N (a) and HEK-IFN4N (b) producing cell lines as a function of puromycin concentration. Both cell lines were cloned using the limiting dilution method, and after 15 days of culture more than 100 clones were screened. To achieve the characterization and study both cell lines as recombinant protein expression hosts, the 6 best producer clones were isolated and amplified. The adherent clones were grown for 7 days in order to construct their growth curves. Cell density and viability were determined every 24 h by trypan-blue exclusion method and the culture supernatant was collected to determine IFN4N and metabolites concentration. The IFN4N production was assessed employing a sandwich ELISA assay developed in our laboratory. Glucose consumption and lactate production were evaluated using specific Reflectoquant® test strips (Merck Millipore) in a RQflex® Reflectometer (Merck Millipore). Levels of amonium in the culture supernatant were determined by the Berthelot reaction. As shown in Table 1, the average specific growth rates of CHO and HEK clones were similar. However, CHO clones reached higher maximum cell densities (between 7.105-1.5.106 cell.ml-1) than HEK clones (between 6.105-9.105 cell.ml-1), probably because of space limitation and higher glucose consumption, since average qgluc of HEK clones was higher (see Table 1). No differences were observed between lactate and ammonium production of both groups of clones. In contrast, specific production rate of IFN4N was higher for the clones derived from the human cell line. Moreover, higher average IFN4N cumulative production for HEK clones was achieved after 7 days of culture (3,494 versus 5,961 ng.ml-1). Table 1 Determination of the specific cell growth rate, specific production rate of lactate, ammonium and IFN4N, and specific consumption rate of glucose of CHO-K1 (a) and HEK 293T (b) clones. a) Clones μ(h-1) qIFN (ng.10-6cell.h-1) qgluc (μg.10-6cell.h-1) qlac (μg.10-6cell.h-1) qamon (nmol.10-6cell.h-1) P4D3 0,0182 ± 0,002 79 ± 8 36 ± 5 40 ± 5 0,027 ± 0,007 P1E9 0,0196 ± 0,002 35 ± 4 24 ± 4 45 ± 5 0,027 ± 0,003 P2A9 0,0249 ± 0,001 41 ± 4 30 ± 2 31 ± 1 0,014 ± 0,004 P1B6 0,0240 ± 0,002 19 ± 3 26 ± 5 49 ± 5 0,013 ± 0,005 P1B7 0,0191 ± 0,002 41 ± 3 32 ± 4 35 ± 2 0,017 ± 0,006 P1B8 0,0277 ± 0,002 42 ± 3 21 ± 3 34 ± 2 0,015 ± 0,004 b) Clones μ(h-1) qIFN (ng.10-6cell.h-1) qgluc (μg.10-6cell.h-1) qlac (μg.10-6cell.h-1) qamon (nmol.10-6cell.h-1) P2A5 0,020 ± 0,001 129 ± 10 56 ± 6 37 ± 4 0,014 ± 0,003 P2C7 0,015 ± 0,002 122 ± 13 62 ± 11 38 ± 3 0,009 ± 0,003 P2G11 0,017 ± 0,002 82 ± 6 47 ± 9 31 ± 3 0,008 ± 0,002 P3B7 0,016 ± 0,002 99 ± 8 55 ± 8 31 ± 3 0,008 ± 0,003 P3H8 0,027 ± 0,001 82 ± 11 46 ± 6 34 ± 3 0,008 ± 0,001 P4B4 0,017 ± 0,002 63 ± 5 61 ± 15 32 ± 3 0,009 ± 0,001 Conclusion CHO and HEK cells were genetically modified to produce IFN4N by using lentiviruses as a tool for the IFN4N gene transfer. Since both cell lines expressed high levels of IFN4N, 6 clones were amplified for an intensive characterization. Culture and production properties of both groups of clones were very different. On the one hand, CHO clones were easy to maintain in culture for a long period of time, reaching higher cell densities than HEK clones. On the other hand, the best specific productivity of IFN4N was achieved employing HEK cells. The behavior of CHO and HEK cells at large scale production should be analyzed in order to select the proper system for the cytokine's production. Wide differences have been observed between the glycosylation profile of the same recombinant therapeutic protein produced in CHO and HEK systems [2]. Considering that glycosylation affects protein bioactivity, stability, pharmacokinetics and immunogenicity, it would be very important to evaluate the characteristics of the IFN4N produced in both hosts to determine their efficacy as therapeutic agents.

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          Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells.

          Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines. Copyright © 2012 Elsevier B.V. All rights reserved.
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            Highly glycosylated human alpha interferon: An insight into a new therapeutic candidate.

            The type I human interferon alpha (hIFN-alpha) family consists of small proteins that exert a multiplicity of biological actions including antiviral, antiproliferative and immunomodulatory effects. However, though administration of recombinant hIFN-alpha2b is the current treatment for chronic hepatitis B and C and for some types of cancers, therapy outcomes have not been completely satisfactory. The short serum half-life and rapid clearance of the cytokine accounts for its low in vivo biological activity. Here we describe and characterize a long-acting rhIFN-alpha2b mutein, 4N-IFN, which has been created by introducing four N-glycosylation sites via site-directed mutagenesis. The hyperglycosylated protein was found to have a 25-fold longer plasma half-life than the non-glycosylated rhIFN-alpha2b, even greater than the commercial pegylated derivative Intron-A PEG. In addition, glycosylation increased the in vitro stability of the mutein against serum protease inactivation. Interestingly, despite its lower in vitro activity, 4N-IFN showed a markedly enhanced in vivo antitumor activity in human prostate carcinoma implanted in nude mice. MALDI-TOF MS and HPAEC-PAD carbohydrate analyses revealed the presence of high amounts of tetrasialylated (40%) and trisialylated (28%) N-glycan structures, which are consequently responsible for the improved characteristics of the cytokine, making 4N-IFN a new therapeutic candidate for viral and malignant diseases. Copyright 2010 Elsevier B.V. All rights reserved.
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              Author and article information

              Contributors
              Conference
              BMC Proc
              BMC Proc
              BMC Proceedings
              BioMed Central
              1753-6561
              2013
              4 December 2013
              : 7
              : Suppl 6
              : P33
              1753-6561-7-S6-P33
              10.1186/1753-6561-7-S6-P33
              3981635
              Copyright © 2013 Gugliotta et al.; licensee BioMed Central Ltd.

              This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

              23rd European Society for Animal Cell Technology (ESACT) Meeting: Better Cells for Better Health
              Lille, France
              23-26 June 2013
              Categories
              Poster Presentation

              Medicine

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