Process for the construction of an animal cell line for the production of human beta-interferon

A method for producing IFN-beta in an animal host cell is described. The IFN-beta isolated from the cell supernatant in high yield has a specific activity of approx. 3.8 x 10⁸ IU / mg protein. Biological and immunological tests show that the isolated IFN-beta is largely identical to the natural IFN-beta. The IFN-beta produced by the process described is 95% glycosylated, the structure and sequence of the glycosylation largely matching that of natural IFN-beta, but without being identical to it.

Interferons are a group of antiviral polypeptides that are formed by the affected cells as a result of contact with exogenous inducers (e.g. viruses, nucleic acids, certain antigens). A subgroup is formed by the beta interferons (IFN-beta), which are mainly formed by fibroblasts, Havell et al. (1972) and Stewart II (1979). So far, 2 species of beta-interferons are known which are similar due to their immunological properties, so that monoclonal, neutralizing antibodies could be isolated, which both inactivate both interferons s. Zilberstein et al. (1985). In contrast, there is no cross-hybridization of IFN-beta-2 mRNA with samples of IFN-beta-1 cDNA in RNA-gel blot hybridization experiments and vice versa, Sehgal et al. (1980).

IFN-beta-1 from human diploid fibroblasts (FS-4), hereinafter referred to as IFN-beta, has been in clinical use for some time. It was approved by the Federal Health Office in 1983 for the treatment of serious life-threatening viral infections. Due to its effectiveness and the lack of other broadly effective antivirals, it has proven to be the drug of choice in many cases.

However, since the production on the basis of normal, non-transformed diploid fibroblasts requires the use of rare and expensive raw materials and the use of costly cell substrates, and since the rationalization of the process sequence has narrow limits, the high price of the drug severely limits the clinical use.

This situation stimulated the search for alternative production methods very early on and was therefore one of the most important drivers for the development of modern genetic engineering processes. The introduction of the gene for human IFN-beta into heterologous host cell systems was the only real alternative to classic optimization methods, since the chances of success seem to be limited due to physiological barriers.

Of the three principally available groups of host cell systems, prokaryotes, lower and higher eukaryotes, the bacterial host system Escherichia coli (E.coli) was initially preferred due to the state of the art and the extremely low production costs.

Despite the high expectations placed in this production system, it has so far not been possible to process the large quantities of crude IFN-beta that can be produced with this method on an industrial scale, Taniguchi et al. (1980) and Goeddel ( 1980). The main reason for these difficulties is that the raw material is present in denatured form as inclusion bodies in the host cell. Although this enables efficient separation of components of the host cell, the yields of clinically usable material have so far been very low due to the problems in the further processing (poor solubility, occurrence of incorrect sulfur bridge bonds). Lawn et al. On the partial lack of effectiveness, which is probably due to conformational changes due to denaturation or improperly formed intrachenaric sulfur bridges. (1981), comes the fact that the product, unlike the naturally occurring form, is not glycosylated.

Against this background, there was an increasing need to advance the development of eukaryotic expression systems, which also allow heterologous gene products with a more complex structure to be produced by correct "processing" in largely authentic form. Systems of this type have been described by a number of authors over time, Reyes et al. (1982), Mitrani-Rosenbaum et al. (1983), Smith et al. (1983), McCormick et al. (1984), Chernajovsky et al. (1984), Fukunaga et al. (1984), Page et al. (1985).

Construction principle

In the following, the construction of a new cell line BIC using genetic engineering methods is described, which makes it possible to produce large amounts of a native product that is largely identical to the natural substance at significantly lower costs than conventional methods.

Host cell line

The host cell line was that of Urlaub et al. (1980) described DHFR^--Mutant of the permanent CHO line (chinese hamster ovary) selected.

Vectors

Interferon gene

The plasmid vector pBR 13, which contains the genomic IFN-beta gene, served as the starting material. A defined section of this genomic DNA was generated according to the methods described by Maniatis et al. (1982) shortened methods and recombined with an already known vector (pSVd2-3).

Promoter

Another important step was the selection of a suitable promoter to regulate expression. In contrast to the possibility of using inducible promoters, Hauser et al. (1982) and Brinster et al. (1982), was that of Mosthaf et al. (1985) described strong constitutive promoter of SV 40, which also contains an enhancer region. The company's own disadvantageous experience with induction-dependent production processes (negative feedback, cytotoxicity) on the one hand and the possibilities that permanent fermentation processes offer for even adherent cell cultures on the other hand led to this decision. The construct described in detail below was called pSVIFNAsu.

selection

For selection and amplification in the direction of a highly expressing cell line, the DHFR gene was introduced separately in a second plasmid pAdD26SV (A) -3. The latter enables the selection for IFN-beta expression through the interaction of one's own adenopromotor with the enhancer region of the SV 40 promoter (see above). In the present construct, as expected, the mutually transfected DNA was integrated in close proximity.

Transfection

The expression vector described above, which contained regulatory sequences and the structural gene, and the selection plasmid, which repairs the deficiency of the cell line, were transfected together in an experimentally determined optimal mixing ratio according to a calcium phosphate precipitation method.

Amplification, cloning and cell bank

Interferon-expressing clones from the transfection approach were used with a first by Kaufman et al. (1985), the method described was amplified over several stages of the methotrexate concentration without intermediate selection to high-producing colonies, then cloned and obtained via serial passages a sufficient number of cells, which were stored as a stem cell bank in liquid nitrogen according to the usual methods of preservation.

Results

Raw material production

The cell line obtained in the described way with the laboratory designation BIC 8622 (BIC, ECACC entry no. 87040301) secretes after confluence in conventional cell culture medium (modified Eagle's MEM with Earle's salts) supplemented with 1-5% NCS or FCS in stationary culture in Multitrays (NUNC) as in the fermenter on microcarriers (Cytodex III, Pharmacia) constitutively between 0.4 and 1.6 * 10⁹ International units of IFN-beta per day in one liter of culture supernatant.

enrichment

Enrichment takes place via adsorption on a sulfopropyl cation exchanger and subsequent immunosorption on anti-IFN-beta-Sepharose (Celltech, Slough). Since this immunosorption matrix contains monoclonal antibodies against native fibroblast interferon, the adsorption and desorption under the conditions optimized by the manufacturer for the immune binding reaction is an indication of the identity of the synthesized molecule, which could be quantified using an ELISA developed using the same antibodies. A further purification step is followed by FPLC gel filtration to remove accompanying substances of low and higher molecular weight.

characterization

Biological effects:

The bioassay developed for the detection of natural IFN-beta from FS-4 cells, which exploits the antiviral activity by quantifying the inhibition of the cytopathic effect of murine encephalomyocarditis virus (EMCV) on an indicator cell line (FS-4), modified Havell et al. (1972) can also be used without restriction for the detection of IFN-beta from BIC cells. The results of this test and Lowry et al. (1951) determined specific activity corresponds to 2-3 * 10⁸ IE within the measurement accuracy of the data determined for FS-4 interferon.

Immunological characterization:

In addition to immunoaffinity chromatography, which was used to enrich the IFN-beta from BIC, the newly developed ELISA and immunoblotting techniques were used to demonstrate extensive agreement in the molecular properties of the natural and the recombinant IFN-beta. The antibodies used are monoclonal from mouse hybridomas (MAK B0-2, Celltech) and polyclonal goat IgG (Rega Institute, Leuwen).

Protein chemical characterization:

The data determined using immunological methods were supplemented by amino acid analysis and sequencing of 15 N-terminal amino acids. No differences between natural, Knight et al. (1980) and recombinant IFN-beta. Examination of the carbohydrate content revealed a largely uniform glycosylation for the recombinant IFN-beta, while the natural IFN-beta showed carbohydrate heterogeneity.

Experimental part

<tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row rowsep="1"><entry namest="col1" nameend="col2" align="left">Materials:</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">E.coli K12 DH 1 (DSM 4079) CHO DUK DHFR-BF (ECACC input) No. 87041401)</entry><entry namest="col2" nameend="col2" align="left">This cell line is the result of mutagenesis and selection for the absence of dihydrofolate reductase from the CHO-K1 line (ATCC CCL 61), Urlaub et al. (1980).</entry></row><row><entry namest="col1" nameend="col1" align="left">pBR 13: (DSM 4074P)</entry><entry namest="col2" nameend="col2" align="left">This plasmid is a derivative of the cloning plasmid pBR 325, Bolivar et al. (1977), in whose only Eco RI interface a 1.83 kb DNA fragment is inserted. This fragment is contained in the cosmid pCos IFN-beta (Gross et al., 1981), which in turn is part of a cosmid library of human placenta DNA.</entry></row><row><entry namest="col1" nameend="col1" align="left">pSVd2-3: (DSM 4075P)</entry><entry namest="col2" nameend="col2" align="left">This plasmid is a derivative of the cloning vector pAT 153, which sequences from the eukaryotic DNA virus SV 40, Fiers et al. (1978) contains. The starting plasmid for the construction of the corresponding vector plasmids was a construction for the expression of the dihydrofolate reductase gene of the mouse, Subramani et al. (1981). The modification of the expression vector pSV was used by Dr. D. Huylebroeck and is essentially in the work of Fransen et al. (1985).</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">pAdD26SV (A) -3: (DSM 4076P)</entry><entry namest="col2" nameend="col2" align="left">Plasmid derivative resulting from the deletion of a 1.1 kb DNA fragment from the cloning vector pBR 322, Lusky et al. (1981). The plasmid also contains the transcription initiation signal (adenovirus major late promoter) from the eukaryotic DNA virus Adeno 2 as well as the cDNA of the dihydrofolate reductase gene from the mouse, the polyadenylation signal and a 200 bp fragment with the origin of the replication from SV 40 , (Kaufman et al. 1982a).</entry></row></tbody></tgroup></table></tables>

Expression system

The expression system used is based on a cell line from ovary cells of the Chinese dwarf hamster (CHO, ATCC CCL 61 CHO-K1), which were made deficient for the enzyme dihydrofolate reductase by mutagenesis, Urlaub et al. (1980). By incubating a calcium phosphate precipitate of DNA with these cells, it is possible to achieve the uptake of this DNA into the cells at a very low frequency, Graham et al. (1973). The DNA thus introduced into the cells is usually linked and built into the genome of the cell in question. There, it is increased or changed exclusively in the course of cellular DNA replication or through recombination processes taking place in the cell genome, so it behaves like an integral natural component of the cellular genome. In the special case, an expression plasmid for the expression of the human IFN-beta gene and, by offering in a mixture, a plasmid for the expression of the cDNA dihydrofolate reductase gene from the mouse was introduced into the CHO cells. The status of the expression plasmid can be determined using the so-called Southern blot technique. For this purpose, the cellular DNA is cleaved with restriction endonucleases and separated on an agarose gel in an electrical field. The DNA fragments are then bound to a nylon filter membrane and detected by DNA-DNA hybridization with radioactively labeled plasmid DNA.

Isolation of the interferon beta gene

The plasmid pBR 13 served as the starting plasmid for the isolation of DNA restriction fragments with the IFN-beta gene. To produce this plasmid, the mRNA coding for IFN-beta from FS 4 fibroblasts was originally translated into cDNA and hybridized with complementary genomic DNA the cosmid pCOSIFN-beta containing the IFN-beta gene isolated from a gene bank (human placenta). By cleaving the plasmid pBR 13 with the restriction endonuclease Eco RI, the fragment with the IFN-beta gene was first released from pCosIFNbeta and then after isolation thereof by further cleavage with the restriction endonucleases Hinc II, or Nco I and Hind III, smaller pieces of DNA the IFN-beta gene.

Construction of the expression plasmids

An expression vector called pSVd2-3 was used to construct the plasmid for expression of human IFN-beta. The functional components of this vector ensure autonomous DNA replication in E. coli and the selection of E. coli cells using ampicillin (origin of the replication of the transfer-negative plasmid pAT 153 and the β-lactamase gene). The components which are intended for a function in eukaryotic cells consist of the promoter-enhancer region of the early genes of the SV 40 virus for initiating the transcription of the downstream genes and of the polyadenylation signal of the SV 40 virus for attaching a polyA sequence to the transcribed one mRNA if the gene used should lack such a signal sequence. Between the signal for transcription initiation and the polyadenylation sequence there are several synthetically generated recognition and cleavage sequences for restriction endonucleases for inserting DNA to be expressed. The cleavage site of the nuclease Xba I was chosen for cloning the human IFN-beta DNA into this vector. To do this, the vector was cleaved with Xba I. An Eco RI fragment with IFN-beta gene was first isolated from plasmid pBR 13 and an Ncol-Hind III subfragment with IFN-beta gene was further isolated from this. The overhanging single-stranded DNA ends resulting from the cleavage with the restriction endonucleases were filled in by incubation with nucleoside triphosphates and the DNA polymer assel from E. coli. Synthetically produced oligonucleotides with the recognition sequence of the restriction endonuclease Xba I were then attached enzymatically to the smooth DNA ends of the fragment thus produced. Excess oligonucleotides were removed by incubation with the nuclease Xba I and thereby overhanging single strand ends were created which were complementary to those of the vector DNA. The vector DNA and the IFN-beta DNA were combined enzymatically and in E. coli cells introduced. The cells with IFN-beta DNA were identified by DNA-DNA hybridization and used to purify larger amounts of plasmid DNA. This plasmid DNA was characterized by cleavage with various restriction endonucleases and that plasmid was selected for the further procedure, and was designated as pSV IPN Nco (DSM 4077P), which met the expectations. The plasmid pSV IFN Nco was then partially cleaved with the enzyme Xba I and the cleavage pieces were separated by gel electrophoresis. The band corresponding to the linear plasmid was cut out after staining with the fluorescent dye ethidium bromide and visualized under UV light and the DNA isolated. The isolated DNA was then incubated with the nuclease Asu II and then closed enzymatically to form a ring. This procedure resulted in the deletion of an Xba I - Asu II fragment from the original expression plasmid pSV IFN Nco. The expression plasmid produced in this way was called pSV IFN Asu (DSM 4078P) and was used to transfect a dihydrofolate reductase deficient CHO cell line. Cells which have been transfected with this expression plasmid pSV IFN Asu and have integrated its DNA into the cell genome are induced to form an mRNA , which consists of a 60 nucleotide long section of SV 40 specific sequences and attached almost the authentic IFN-beta mRNA. This mRNA is translated by the cell's protein synthesis apparatus into an IFN-beta protein which corresponds in its amino-terminal region and in its amino acid composition to the natural IFN-beta protein from human fibroblast cells and in contrast to the proteins produced in E. coli is glycosylated.

The primary structure of the mRNA, which is formed in eukaryotic cells which have integrated this plasmid into their genome, can be derived from the DNA species used and the construction of the expression plasmid pSV IFN Asu. The structure of the expression plasmid is shown in Fig. II with a detailed expression of the relevant interfaces. A translation of the nucleotide sequence using the genetic code into an amino acid sequence gives the primary structure, the authentic IFN-beta from human cells, Tavernier et al. (1984).

Transfection

The expression vector DNA was introduced into the CHO cell line essentially by a method by Graham et al. (1973) and modified by Wigler (1979). In principle, this method offers the cells DNA that has been precipitated from a solution with CaPO4 as a coprecipitate. Probably through phagocytosis, this DNA is then taken up by the cells and usually strung together via mechanisms that have not yet been elucidated and integrated into the cellular genome. This process is called transfection of eukaryotic cells with DNA. By co-precipitating a vector for the expression of the enzyme dihydrofolate reductase with the actual expression vector, when using dihydrofolate-deficient CHO cells, those cells can be selected after the transfection process that have taken up DNA and integrated it into their genome. This is done by culturing the cells in a cell culture medium that lacks building blocks for nucleic acid synthesis.

Amplification and selection

The culture supernatants of the cells transfected and selected in this way were checked for beta-interferon activity. The clones positive in this test were subjected to a process which aimed to selectively multiply the DNA introduced into the cells and to achieve an increase in beta-interferon production by increasing the copy number of the interferon genes. This is basically possible according to the gene dose effect. The method used for this purpose provides for a selection of the beta-interferon-producing cells in culture medium which contains the enzyme inhibitor (+) amethopterin. This inhibitor inhibits the enzyme dihydrofolate reductase, the gene of which had been introduced into the CHO cells by the transfection, depending on the concentration. As a result of this inhibition, cells which have increased the gene dose of the dihydrofolate reductase gene by increasing the corresponding DNA have a growth advantage. Since larger sections are usually involved in such DNA duplication processes and the transfected DNA species are usually integrated into the genome of the cells, the gene dose of the beta-interferon gene is increased at the same time. By gradually increasing the (+) amethopterin concentration in the culture medium and intervening selection and expansion phases, a cell mixture was produced which releases a large amount of beta-interferon into the culture medium. Pure lines of cells which also release large amounts of beta-interferon into the culture medium were produced from this mixture by dilute sowing of the cells in the culture vessels and isolation of the cell colonies with the aid of metal cylinders.

A manufacturer cell bank (= production cell bank) was set up in accordance with the relevant guidelines of these clones designated as "BIC" cells and aliquots of these cells were obtained from the "Public Health Laboratory Service, European Collection of Animal Cell Cultures (ECACC)" with the entry no. 87040301 deposited.

enrichment

From a confluent stationary culture of BIC, which was taken from the production cell bank, 15 I culture supernatants with an average interferon content of 225,000 IU / ml were pooled by repeated harvesting in a 24-hour cycle and applied together to a cation exchanger (sulfopropyl). In 175 ml of eluate there were 3 * 10⁹ IE included, which corresponds to a yield of 83%. The subsequent immunosorption on BETA RESOLUTE (R) (Celltech) resulted in an elution volume of 112 ml, in which 2.2 * 10⁹ IE still 72% of the interferon with a specific activity of 3.8 x 10⁸ IU / mg protein were included. The final gel filtration gave an overall yield of 48%.

Analytics

Biological and immunological characterization

Highly purified preparations of recombinant BIC-beta-IFN and natural FS 4-beta-IFN were tested for their interferon content in the antiviral bioassay. In parallel, a Lowry protein content determination was carried out. The specific activity for the natural material was about 2.7-3 * 10⁸ International units (IU) per milligram of protein and an activity of 3 - 4 x 10 for the recombinant material⁸ IU / mg. These values ​​correspond to the data for natural interferon known from the literature. Due to the relatively high measurement inaccuracy of the bioassay, a more precise determination of the spec. State of the art activity cannot currently be achieved.

The starting material and the enriched and highly purified fractions were subjected to an enzyme-linked immunoassay (ELISA) in addition to the antiviral bioassay.

For this test, the plastic surface of microtiter plates is coated with goat polyclonal anti-IFN-beta antibodies. A dilution of the sample to be determined is then applied. The beta IFN now binds to the immobilized antibodies. In the next step, this complex is allowed to react with a solution of mouse anti-IFN-beta monoclonal antibodies. The resulting complex is then incubated with anti-mouse IgG antibodies that were previously conjugated with horseradish peroxidase. After removal of the unbound antibodies, the amount of bound peroxidase and thus also the amount of bound beta-IFN can be determined by a color reaction.

This test has been validated for natural beta-IFN derived from FS 4 fibroblasts. For this purpose, dilution series of samples with known interferon content were checked together with aliquots of the international beta-IFN standard (NIH, G-023-902-527). There was always a strict correlation between the bioassay and the ELISA.

To the same extent, the enriched and highly purified IFN preparations obtained from the culture supernatants of BIC cells in the ELISA corresponded to the values ​​which were determined in parallel in the antiviral bioassay. In all cases, aliquots of the international standard or preparations calibrated on them were used for standardization. It could thus be shown that the antigenic properties relevant for the ELISA between natural and recombinant beta-IFN are identical.

Sequence / amino acid composition

The recombinant IFN-beta produced and purified by the described method was analyzed with regard to its amino acid composition and its amino-terminal sequence.

The partial sequencing up to the 15th amino acid was carried out by means of Edman degradation in an automatic gas phase sequencer (Applied Biosystems, type 470 A). The resulting phenylhydantoin derivatives were separated on a PTH-C18 matrix and then detected. The result agrees with that of Lawn et al. (1981), Ohno et al. (1981) and Derynck et al. (1980) published data. When analyzing the amino acid composition, in which the PTH amino acids were subjected to the same separation method after hydrolysis, the results described by Knight et al. (1980) values ​​determined for FS-4 can also be confirmed for IFN-beta from CHO.

Glycosylation

The polypeptide obtained after deglycosylation or after inhibition of glycosylation with tunicamycin had the same electrophoretic properties under denaturing and non-denaturing conditions as the IFN-beta obtained in the same way from FS-4. The oligosaccharides released by glycopeptidase F were subjected to methylation analysis and FAB mass spectrometry after sequential degradation by exoglycosidase. It was found that 95 ± 5% of the carbohydrate side chains were biantennary complex type and had the following structure:<chemistry id="chem0001" num="0001"><img file="EP0287075B2_D0001.tif" /></chemistry>

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