WO1994025599A1 - Mammal cell lines and method of obtaining glycoproteins - Google Patents

Abstract

The invention concerns mammal cell lines, in particular hamster cell lines, which express in stable fashion the glycoproteins gp 250 (220), 350 and 250 (220)/350 as Epstein-Barr virus antigens, plus a method of obtaining these glycoproteins from cells cultivated in a protein-free medium.

Description

 Mammalian cell lines and methods for glycoprotein recovery

The Epstein-Barr virus is the causative agent of infectious mononucleosis in humans, one of the most common infectious diseases in young adults.

Over 90% of the adult population has had an Epstein-Barr virus infection. These EBV-positive people can later develop diseases such as nasopharyngeal carcinomas, B-cell lymphomas, T-cell lymphomas, certain forms of Hodgkin's lymphoma, and possibly other malignant diseases. Chronic forms of the course and, in rare cases, even acute fatal cases are described directly in connection with the primary infection or later.

Since the infection with Epstein-Barr virus is clinically normal in young children and because vaccination with recombinant vaccine viruses which contain the Epstein-Barr virus membrane antigen gp 250/350 (BLLF-1 reading frame, Baer et al., 1984) express a protection against infection or disease (Gu et al., 1993), it can be assumed that vaccination against Epstein-Barr virus-corrected diseases is possible.

Because of the possible side effects of live vaccines, the development of pure protein vaccines is therefore an important goal. Since the Epstein-Barr virus membrane protein gp 250/350 (BLLF-1) is very strongly glycolized and this post-translational modification is an important component of the correct immunogenicity (Emini et al. , 1988), the development of a vaccine based on a glycoprotein that can be produced in eukaryotic cells and based on the gp 250/350 (reading frame BLLF-1 of the Epstein-Barr virus) is a promising route.

The invention relates to mammalian cell lines, in particular hamster cell lines, which stably contain the glycoprotein (gp 250 (220)), the glycoprotein (gp 350) and / or the mixed glycoprotein (gp 250 (220) / (gp) 350 ) as Epstein-Barr virus antigens, especially in a protein-free medium. This prevents the risk of viral or bacterial contamination from media supplements. The invention further relates to a method for obtaining the glycoproteins mentioned.

Hamster cell lines which stably express gp 250 (220), gp 350 and or gp 250 (220) / gp 350 as EBV antigens are known; compare, for example, Motz et al. in Gene, 44 (1986) 353-359 and Vaccines, 87 (1987) 374-379 and Gu Shyan et al. in 100 years blood serum therapy 1890-1990, Halle (Saale), (1991) 93-100. So far, however, no hamster cell lines have become known with which the glycoproteins mentioned can be stably expressed. According to Motz et al. in Vaccines, 87 (1987) 374-379, 376, the integration of the viral foreign glycoproteins in CHO cell membranes can be toxic to a certain extent.

According to one embodiment of the invention, by using a targeted genetic strategy, a hamster cell line has now been made available which stably expresses and secretes the glycoproteins mentioned and is obtainable by:

the cell line is infected by transfection with a vector expressing the glycoproteins mentioned, but which does not express the membrane anchor (Motz et al., 1987), the vector comprising a selection marker which the cell never lacks, the cell line is cultivated and selected with the aid of the selection marker for cells which, after omitting the selection factor (inhibitor), stably express and secrete the glycoproteins.

According to a further embodiment, a hamster cell line was also made available through the use of a method which stably expresses the glycoproteins mentioned in a protein-free medium and is obtainable by:

(A) starts from a hamster cell line (host cell line) which lacks a selection marker which a vector to be integrated chromosomally later according to (A) (d) or a vector integrated according to (B) (recombinant cell line) has,

(a) the cells are cultivated in a serum-containing medium and thereby adhere to the substrate and to one another,

(b) repeatedly exchanges a part of the used medium, gradually reduces the serum content of the medium during the exchange and finally cultivates it serum-free and does not actively remove the adherence of the cells and, if necessary, does not actively form and detach spheroids from cells, separating the detached cell spheroids , cultivated in suspension with gentle movement and selected for cells that grow in small aggregates or individually and

(c) finally subjecting the selected cells to the procedural measures of claim 1, or

(B) that a hamster cell line according to claim 1 is subjected to process steps (A) (a) to (A) (b).

The starting cell line or the host cell line is therefore grown in a culture medium containing serum, for example in MEM alpha ^" or a ^" + 5% FCS, since this cell line cannot yet be transferred into media and cultured in media which are free of serum. Examples of such culture media are DIF 1000, RPMI 1640, ASF 103 and HDB. In order to achieve growth in protein-free culture medium, 3 aspects have to be mentioned.

1. One uses the adherence during the serum weaning of the culture, whereby one works for example in culture bottles. This results in positive growth stimulation. In addition, old used medium and dead cells as well as lysis products can be separated easily and very gently (without centrifugation).

2. The old medium is repeatedly only partially replaced in order to guarantee good self-conditioning of the medium during the thinning of the serum.

3. Long-term cultivation of the cells is carried out with gentle mixing, for example in spinner bottles in suspension culture with formation of spheroids. By selective selection for reduced adherence efforts, cells can be isolated that grow in small aggregates (spheroids) or individually. First you will separate large spheroids. Then the supernatant is fractionally centrifuged at low g-numbers and the separated small spheroids and single cells are used for further passage.

Growth is possible in the following protein-free medium using the strategy set out in claim 2. The medium is referred to below as SMIF (Scharfenberg's Modified Iscove's-F12 Medium). It is a well-enriched medium, consisting of an approximate 1: 1 mixture of Iscove's-Modified-Dulbecco's medium with Urine’s F12 nutrient solution (IF), which also contains, for example, putrescine (for example 1, 2 micromoles 1) and L-hydroxyproline (for example 153 micromoles 1 ^"1 ) (SMIF1) can be added. In addition, when growing in suspension, chelators such as aurintricarboxylic acid (ATA, for example 3 micromoles 1 ^{" 1} ) (Bertheussen, 1993), EDTA (at Example 4/3 micromole 1) and citric acid (for example 40 micromole 1), for complexing divalent ions and for better availability of inorganic iron as a replacement for the protein component customary in serum-free media to add transferrin (SMIF2) .

According to a special embodiment of the invention, hamster cell lines can be obtained by using a Chinese hamster ovary cell line (CHO), for example CHO Kl = ATTC CCL 61, or a baby hamster kidney cell line (BHK). , for example BHK 21C13 = ATCC CCL 10.

If, according to the invention, hamster cell lines with generation times in the range of, for example, 15 to 40 and in particular 20 to 30 hours can be made available, with which the glycoproteins mentioned can be stably expressed, this performance is to be regarded as inventive in that it has so far been it has not yet been possible to cultivate hamster cell lines in protein-free medium without targeted genetic manipulation (genetic engineering). Until now, it was believed that one had to prepare sucker cell lines for growth in protein-free medium for the expression of desired gene products by gene manipulation (NSO cell line, Hassel et al., 1992).

The person skilled in the art can adhere to the relevant prior art for selection markers and inhibitors. An example of a suitable selection marker is the dihydrofolate reductase gene (DHFR gene) and an example of a suitable inhibitor is methotrexate (MTX) (Motz et al., 1987).

Finally, a special embodiment of the invention is the hamster cell line CHO pMDIIIGPTR-PFC6 = DSM ACC 2121.

Gp 250 (220), gp 350 and / or gp 250 (220) / gp 350 can be obtained according to the invention by: (a) cultivating a cell line according to the invention and having the desired glycoproteins expressed,

(b) the cells are separated from the culture medium, in particular by microfiltration,

(c) then the clear raw supernatant is concentrated and purified by ultrafiltration, in particular by crossflow UF,

(d) then using an anion exchange column,

(e) ultrafiltration and

(f) a gel filtration is purified and the resulting fractions of pure glycoproteins (gp) are recovered.

The glycoprotein can be obtained natively and glycosylated by the process according to the invention. This gives them their natural antigenic potential. The vaccine obtained in this way accordingly has antigenic properties which correspond to those of the corresponding proteins of the active EB virus. The vaccine obtained is thus a safe vaccine with a high potential of antigenic protection without pathogenic potential. The recombinant vaccine is thus superior to an inactivated or weakened vaccine in terms of safety, since neither a potential for reversing weakened viruses nor the residual risk and the Toxicology of inactivated viruses are to be feared.

Example 1: Recovery of CHO pMDIIIGPTR-PFC6

A clone derived from the cell line CHO Kl = ATCC CCL61, which lacks the dihydrofolate reductase gene (dhfr-1), with the plasmid pMDIIIGP according to Motz et al. in Gene, 44 (1986) 353-359, Motz et al. in Gene, 58, (1987) 149-154 and Vaccines, 87 (1987) 374-379. The cells were cultivated and selected with the aid of methotrexate (MTX) for clones which stably expressed gp 250/350 even after the inhibitor had been omitted. A clone with stable expression of gp 250/350 was designated as CHO C6. Example 2: Cultivation of CHO C6 in protein-free medium

One worked under standard cell culture conditions. CHO C6 was cultured in IF medium containing 5% fetal calf serum (FCS) on 1 to 2 large culture bottles (185 cm ² , about 40 to 50 ml volume; Falcon) until absolute cell confluence, with multilayers already forming in some cases . The subsequent thinning of FCS was carried out in stages over an FCS content of 1, 0.2, 0.04 and 0.008%, by always 4/5 of the old medium including living and dead free cells and lysis products by fresh, preheated SMIF1 have been replaced. If the culture formed free spheroids even at low FCS contents, these could settle before the medium was removed. The point in time of the exchange was determined by the vitality and the state of the culture and was determined on a case-by-case basis, at the latest when the culture supernatant contained insufficient amounts of nutrients. A good guideline for a change is a glucose content of about 0.7 to 1 g / 1. At the latest after a week, a smaller part was exchanged to supplement decayed nutrient substances.

When the medium containing 0.008% FCS was exhausted, the entire medium was replaced with fresh protein-free preheated SMIF1 and further cultivated in the culture bottles. If sufficient spheroids had already formed and detached under these conditions, these spheroids were transferred into a small spinner culture bottle (40 ml minimum volume) and in about 40 ml SMIF1 (1/4 old conditioned plus 3/4 fresh medium) 40 rpm cultivated. If the cells did not detach, the cell lawn had to be isolated enzymatically from the substrate, as is normal for the passage of adherent cells. The cells, which clump very easily in this state, were washed free of protease twice by SMIF1 medium after detachment, before the cells len could also be transferred into a small spinner bottle.

The cells were cultivated in the spinner bottle until generation times of less than 40 hours were obtained and the culture grew in small spheroids. Passenging was carried out with a guideline value of about 0.5 to 1 g of residual glucose / 1 in the medium. Small spheroids and single cells were targeted. For this, the cell culture suspension was left to stand for a short time so that large spheroids could settle, after which the supernatant (100 g) was centrifuged. The cells in the pellet were then transferred to the next passage. The culture medium was conditioned by adding 20 to 30% by volume of medium from the previous passage, which had previously been freed from dead cells and cell fragments by means of filtration or centrifugation.

The entire process took longer than two months for two attempts to adapt.

Example 3: Processing and purification of the recombinant proteins from culture supernatants

I. Cross-flow ultrafiltration with subsequent buffering

For this, the microfiltered harvests of fermentations in protein-free SMIF1 or SMIF2 were used, namely from continuously perfused culture up to the 2-1 scale or from batch culture in a 10-1 airlift fermenter. Filter cartridges with a nominal cut-off of 100 kD (Milipore or Filtron) were used for the filtration. Guide values during cross-flow ultrafiltration with a relatively free choice were: Transmembrane pressure not above approx. 1 bar; about 1/3 of the crude supernatant to be filtered which was in the circuit was separated from the volume flow as filtrate; with a filter cassette and a suction capacity of, for example, 8 l / min, this corresponded to about 2-3 l of filtrate formation; lower pumping capacities were used during the buffer exchange.

In general, the following stages were provided, it being possible to dispense with the first two steps, which, however, can increase the yield.

1. The total harvest volume was determined and about 15% by volume of filtrate (based on the volume flows under normal filtration conditions) was produced in order to condition the membrane, ie. H. to set a constant effective filtration exclusion limit by covering and polarizing the membrane.

2. This first filtrate was returned to the retentate.

3. The actual cross-flow ultrafiltration was carried out until the retentate was reduced to about 1 / 50th of the starting volume. However, an even stronger concentration is possible, especially by using a protein-free medium.

4. The buffer change and washing process of the primary retentate took place after the final volume was reached. For this purpose, the retentate was filled three to four times to twice the volume (including the dead volume of the pump and UF system) with phosphate buffer (20 mM of pH 7) and each time again concentrated to the minimum retentate volume and for the further processing planned. This step was carried out to remove low molecular weight foreign proteins and to reduce the ionic strength for the subsequent purification step. II. Purification with an anion exchange column

The following equipment was chosen.

Column material: Q-Sepharose High Performance (a BioProcess material from Pharmacia)

Buffer system: Buffer A: 20 mM phosphate buffer (pH 5.1)

Buffer B: 20 mM phosphate buffer (pH 5.1) with 1 M NaCl

Scale: for example small scale with FPLC system and column HR 5/5 with 1 ml bed volume (Pharmacia); Scale-up possible.

Flow: constant 5.1 cm / min corresponding to 1 ml / min with HR 5/5

The ultrafiltration retentate was diluted to a conductivity of less than 3 S with MiliQ process water and adjusted to a pH of 5.1 with HC1 (for example first to pH 4.2 in order to precipitate a larger proportion of foreign proteins and then to ¬ back to pH 5.1). The resulting turbidity was precipitated by centrifugation. Then the clear supernatant was added via an FPLC pump with the addition of a 5 percent. Portion of Buffer B applied to the column to elute first foreign protein. The maximum loading was about 8 mg total protein / ml gel. After the application, the protein was eluted through a gradient up to 0.4 M NaCl (increase over 22 column volumes).

The desired gp 250/350 protein eluted between 0.15 and 0.35 M NaCl content, the exact selection of the fractions to be purified further being decided after polyacrylamide gel electrophoresis (PAGE). A protein application and a separation could also be achieved without the addition of buffer B and also at other pH values (eg pH 7). The low pH and the Zudo- Buffer B during the application increases the performance in relation to the gp proteins in chromatography.

If phosphate buffer was used, there was no need for a re-buffering step before the subsequent work-up steps. However, other buffer systems, such as citrate buffers, are also possible.

III. Ultrafiltration

The selected and combined protein fractions from step II were concentrated by a factor of about 10 to 20 by ultrafiltration before application to a gel filtration column. The final protein concentrations were, for example, in the range of 1 to 2 mg / ml. Minicon CS15 chambers (Amicon) were used for the ultrafiltration. (For larger solution volumes, think of correspondingly larger filtration devices with larger capacities, for example stirred cells or crossflow ultrafiltration modules.)

IV. Purification by gel filtration

Column material: finished columns HR 10/30 with Superose 6 (Pharmacia); for a scale up, Sephacryl S-300 High Resolution and Sephacryl S-400 High Resolution in an XK 26/100 column (Pharma¬ cia) were used.

Buffer system: Isocratic 100 or 200 mM NaCl in a 20 mM phosphate buffer (pH 7).

Flow: for example 2-2.5 mm / min After the column was equilibrated, 10 g of protein per ml of gel bed, for example, was applied, which had previously been concentrated (CS15) and freed of turbidity by centrifugation. It was eluted at a flow of about 2.5 mm / min and separated. The elution fractions were combined which, after PAGE and silver staining, contained gp 250 and or gp 350 without visible impurities.

Pure gp 250/350 can be stored in a stable manner at about -20 ° C. in the collected gp fractions of the gel filtration. Overall, about 25-40% of the crude product was obtained purely through the purification process.

Comparative Example 1

Example 2 was repeated with the exception that no cell confluence was permitted in the serum weaning phase and the FCS thinning was carried out with cell adherence removed. In this procedure, no cells that could be cultivated in a protein-free medium could be obtained.

literature

Baer, R. et al. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature, 310 (1984) 207-211.

Bertheussen, K. Growth of cells in a new defined protein-free edium. Cytotechnology, 11 (1993) 219-231

Emini, EA et al. Antigenic analysis of the Epstein-Barr virus major membrane antigen (gp 350/220) expressed in yeast and mammalian cells; implications for the development of a subunit vaccine. Virology, 166 (1988) 387-93. Gu, S. et al. On the first EBV vaccine trial in humans using recombinant vaccina virus expressing the major membrane antigen. Proceedings of the Vth International Symposium on "Epstein-Barr virus and associated Diseases", Annecy, 1992 (1993)

Hassel, T. et al. Stability of recombinant antibodies from glutamine-synthetase amplified CHO and NSO cell lines. In: Animal Cell Technology: Developments, Porcesses and Products (Spier, R.E., Griffiths, J.B., MacDonald, C., eds.) Butterworths-Heinemann, Oxford, (1992) 42-47

Motz, M. et al. Truncated versions of the two major Epstein-Barr viral glycoproteins (gp250 / 350) are secreted by recombinant Chinese hamster ovary cells. Gene, 58: 149-154 (1987)

Claims

Claims 1. mammalian cell line which stably expresses the glycoprotein (gp) 250 (220), the glycoprotein (gp) 350 and / or the mixed glycoprotein (gp) 250 (220) / (gp) 350 as Epstein-Barr virus antigens and that it is obtainable by transfecting the cell line with a vector expressing said glycoproteins (gp), the vector comprising a selection marker which is missing from the cell line, the cell line being cultivated and selected for cells with the aid of the selection marker that stably express the glycoproteins after omitting the selecting factor (inhibitor). 2. Mammalian cell line which stably contains the glycoprotein (gp) 250 (220), the glycoprotein (gp) 350 and / or the mixed glycoprotein (gp) 250 (220) / (gp) 350 as Epstein-Barr virus antigens is expressed in a protein-free medium and is obtainable by (A) starts from a hamster cell line which lacks a selection marker which a vector to be integrated chromosomally later according to (A) (d) or a vector chromosomally integrated according to (B) has, (a) the cells are cultivated in a serum-containing medium and thereby adhere to the substrate and to one another, (b) repeatedly exchanges part of the used medium, gradually reduces the serum content of the medium during the exchange and finally cultivates it serum-free and does not actively cancel the adherence of the cells and optionally forming and detaching spheroids from cells, separating the detached cell spheroids, cultivating them in suspension with gentle movement and selecting for cells that grow in small aggregates or individually and (c) finally subjecting the selected cells to the procedural measures of claim 1 or (B) subjecting a mammalian cell line to process steps (A) (a) to (A) (b). 3. mammalian cell line according to claim 1 or 2, characterized in that as a mammalian cell line a hamster cell line, in particular a Chinese hamster ovary cell line (CHO), for example CHO Kl = ATCC CCL61, or a baby hamster kidney cell line (BHK ), for example BHK 21 C13 = ATCC CCL10. 4. Hamster cell line according to one of the preceding claims, characterized in that the dihy- drofolate reductase gene (DHFR gene) and the inhibitor methotrexate as the selection marker (MTX) used. 5. Hamster cell line CH0-K1 pMDIIIGPTR-PFC6 = DSM ACC 2121. 6. Process for the production of the glycoprotein (gp) 250 (220), the glycoprotein (gp) 350 and / or the mixed protein (gp) 250 (220) / (gp) 250, characterized in that one (a) cultivating a cell line according to one of claims 1 to 5 and expressing the desired glycoproteins and allowing them to be secreted into the medium, (b) the cells are separated from the culture medium, in particular by microfiltration, (c) then concentrated and purified by cross-flow ultrafiltration, (d) then using an anion exchange column, (e) ultrafiltration and (f) a gel filtration is purified and the pure glycoprotein fractions (gp) obtained.