DE4313620A1 - Hamster cell lines and methods for glycoprotein recovery - Google Patents

Description

The invention relates to hamster cell lines that are stable to glyco protein (gp 250 (220)), the glycoprotein (gp 350) and / or the Mixed glycoprotein (gp 250 (220) / gp 350) as Epstein-Barr virus Can express antigens, especially in a protein-free one Medium. The invention further relates to a method for winning tion of the glycoproteins mentioned.

Hamster cell lines that are stable gp 250 (220), gp 350 and / or gp Expressing 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 Shuyan et al. in 100 Years of blood serum therapy 1890-1990, Halle (Saale), (1991) 93-100. However, no hamster cell lines are known yet with which the glycoproteins mentioned stable expri let it smear. According to Motz et al. in Vaccines, 87 (1987) 374-379, 376 the integration of viral foreign glycoproteins in CHO cell membranes can be toxic at a certain density.

According to one embodiment of the invention, it has now been surprising provided a hamster cell line that sta bil expressed the glycoproteins and thereby obtained Lich is that you have hamster cell lines that are stable to the glycoprotein (gp) 250 (220), the glycoprotein (gp) 350 and / or the mixed  Glycoprotein (gp) 250 (220) / (gp) 350 as Epstein-Barr virus Antigens are expressed and can be obtained by

• - The cell line with a said glycoproteins express vector infected by transfection,
• - The vector comprising a selection marker that the cell line missing,
• - Cultivated the cell line and with the help of the selection marker selected for cells that, after omitting the selek tioning factor (inhibitors) stable the glycoproteins express.

According to a further embodiment, it was also surprising provided a hamster cell line that sta bil the glycoproteins mentioned in a protein-free medium is expressed and obtainable in that one

• (A) proceeds from a hamster cell line that is a selection marker is missing, which is added chromosomally according to (A) (d) integrating vector or a chromosomal according to (B) has integrated vector,
• (a) culturing the cells in a serum-containing medium and adheres to the reactor and to one another,
• (b) repeats a portion of the media used exchanges the serum content of the Medium reduced and finally cultivated serum-free And thereby
• - does not actively remove the adherence of the cells and
• - if necessary, form and remove spheroids from cells lets, the detached cell spheroids separated, in Suspen sion cultivated with gentle movement and on cells selected in small aggregates or individually grow and
• (c) Finally, the selected cells follow the procedure subject to measures of claim 1, or
• (B) that a vertebrate cell line according to claim 1 subjects (A) (a) to (A) (b).

The starting cell line is left in a serum-containing kul grow medium, for example in MEM a⁻ + 5% FCS, because do not yet transfer this cell line into media and into media can be cultivated, which are free of serum, for example FCS. Examples of culture media are also DIF 1000, RPMI 1640, ASF 103 and HDB.

To achieve an adaptation to a culture medium containing serum 3 aspects have to be mentioned.

• 1. One uses the adherence during the serum weaning of the culture from, for example, working in culture bottles. This results in positive growth stimulation. Al used medium and dead cells as well as lysis products can be easily and very gently (without centrifugation) split off.
• 2. Old medium is repeatedly only partially replaced in order a good conditioning of the medium during the serum guarantee thinning.
• 3. One sees a long-term cultivation of the cells under scho movement, for example in spinner bottles in Suspen culture for spheroid formation. Through targeted Selek can be based on reduced adherence efforts small aggregates (spheroids) or individually growing cells isolate. First you will separate large spheroids. After that the supernatant from another centrifugation is never at subject to other g-numbers. For another passenger the small spheroids from the pellet sepa rieren.

For example, you can adapt to one medium, the next is called S-IF. It is a well received enriched medium, consisting of a 1: 1 mixture from Isco ve’s-Modified-Dulbecco’s medium with Ham’s-F 12 nutrient solution  (IF), which also contains 160 mg putreszine / l (final concentration for example 240 µg / l) and as an additional ingredient L-hydroxyproline (concentration 20 µg / l) can be added.

According to a special embodiment of the invention, these are Hamster cell lines can be obtained by Hamster ovary cell line (CHO), for example CHO K1 = ATCC CCL61, or from a baby hamster kidney cell line (BAK), for example BHK 21C13 = ATCC CCL10.

If, according to the invention, hamster cell lines with generation time ten in the range of, for example, 15 to 40 and in particular 20 up to 30 h available with whom the named Glycoproteins are stably expressable, so is this achievement to be regarded as inventive in that it has not yet succeeded in serum-free cultivable hamster cell lines without ge aimed to provide genetic engineering. So far it was believed that hamster Cell lines for the expression of desired gene products by gene manipulation must be tailored.

Those skilled in the art can know for selection markers and inhibitors keep the relevant state of the art. An example of one a suitable selection marker is the dihydrofolate reductase gene (DHFR gene) and is an example of a suitable inhibitor Methotrexate (MTX).

Finally, a special embodiment of the invention is Hamster cell line CHO K6 = DSM ACC 2121.

Gp 250 (220), gp 350 and / or gp 250 (220) / gp 350 can be win according to the invention in that

• (a) a cell line according to the invention is cultivated and the desired ones can express glycoproteins,
• (b) the cells are separated from the culture medium, in particular by Mi crofiltration,
• (c) then concentrated and purified by cross-flow UF,  
• (d) then using an anion exchange column,
• (e) ultrafiltration and
• (f) a gel filtration purifies and the resulting glycopro teine (gp) wins, if necessary after fractionation.

According to the method of the invention, the glycopro win a native and glycosylated. With that they own her natural antigenic potential. The one obtained in this way Accordingly, vaccine has antigenic properties that act match the EB virus. The vaccine thus obtained provides a safe vaccine with a high potential for antigenic Protection without pathogenic potential. The recombinant vaccine is thus related to an inactivated or weakened vaccine superior to security since there is no potential for rever weakened viruses, the residual risk and the toxicology inactivated viruses are to be feared.

Example 1: Obtaining CHO K6

It was CHO K1 = ATCC CCL6I, a cell line that lacks the dihydrofo lat reductase gene (dhfr ^-1 ), with the plasmid pMD III GP according to Motz et al. in Gene, 44 (1986) 353-359 and Vaccines 87 (1987) 374-379 by transfection. The cells were cultivated and selected using 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 named CHO K6.

Example 2: Cultivation of CHO K6 in protein-free medium

It worked at 37 C in air with a CO₂ content of 10 to 14% and 100% humidity. CHO K6 was in IF medium with a Content of 5% fetal calf serum (FCS) on 1 to 2 large kul door bottles (185 cm², about 40 to 50 ml volume; Falcon) up to cultivated absolute cell confluence, whereby already partially  Multilayers trained. The subsequent thinning of FCS was carried out gradually over an FCS share of 1, 0.2, 0.04 and 0.008% by always including 4/5 of the old medium free and dead cells and lysis products fresh, preheated S-IF have been replaced. Unless the culture formed free spheroids even at low FCS contents they settle before removing the medium. Point of time of the exchange was due to the vitality and condition of the Culture and was determined on a case-by-case basis, at the latest then when the culture supernatant is too small in amounts of nutrients contained. A good indication for a change is a glucose Ge hold from about 0.7 to 1 g / l. After a week at the latest to a lesser extent replaced to decayed nutrients punch to complete.

When the medium was exhausted with 0.008% FSC, the whole was Medium replaced by fresh serum-free preheated S-IF and further cultivated in the culture bottles. If among them Conditions already formed and detached enough spheroids, these spheroids were placed in a small spinner culture bottle (40 ml minimum volume) and transferred to 40 ml S-IF (¼ old conditioned plus 3/4 fresh medium) at approx. 40 rpm cul activated. If the cells did not detach, the cell line had to be abtrypsinized, i.e. a cell culture treatment under are thrown as he nor for the adherence of adherent cells times is. The cells clumping very easily in this state were freed of protease twice through medium after detachment before the cells also in a small spinner could be transferred.

The cells were cultivated in the spinner until the culture Showed generation times of less than 40 h and in small Spheroids grew. Passengers took place, if only about 0.5 to 1 g of residual glucose / l were present in the medium. There were targeted passage of small spheroids and individual cells. Therefor the cell culture suspension was left to stand for a short time, so that large spheroids could settle and pelletize  the supernatant (100 g). The cells in the pellet were then transferred to the next passage inoculated. The culture medium became condensed tioned by 20 to 30 vol .-% medium of the previous Pas say admit that previously of dead cells and cell debris had been freed by filtration or centrifugation.

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

Example 3: Processing and purification of culture supernatants I. Cross-ultrafiltration with subsequent buffering

For this, the microfiltered harvests of fermentations in serum-free S-IF used, and that from continuous perfume culture up to 2 l scale or from batch in 10 l airlift Fermenter. Filter cartridges were of a nominal size Cut-off limit of 100 kD (Millipore or Filtron) used. Guide values during cross-ultrafiltration at re relatively free choice were:

Transmembrane pressure not over 1 bar;
about 1/3 of the volume flow became filtrate; In a filter cassette, this corresponded to a suction capacity of, for example, 6 l / min, about 2 l of filtrate formation;
low pumping capacity during the buffering.

In general, the following stages have been provided, with the The first two steps are not absolutely necessary, however can increase the yield.

• 1. The total harvest volume was determined and about 15 were produced Vol .-% filtrate (based on the volume flows under normal Filtration conditions) to condition the membrane, i.e. H. a constant effective filtration exclusion limit Set the assignment and polarization of the membrane.  
• 2. The filtrate was returned to the retentate.
• 3. The actual cross ultrafiltration was carried out until the retentate reduced to about 1 / 50th of the initial volume was. However, an even stronger concentration is possible Lich.
• 4. Buffering and washing of the primary retentate was done after the final volume was reached. This was done three to four times the retentate to twice the volume (including the Dead volume of the pump and UF system) with phosphate buffers (20 mM from pH 7) and each time again on the mini male retentate volume concentrated. The filtrate for analysis and possible further processing The buffered and washed retentate is in this Zu stood at about -20 ° C freezable. It was for the further Refurbishment provided.

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); sacle-up possible.

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

The ultrafiltration retentate was made up with MiliQ process water a conductivity of less than 3 mS and diluted with HCl adjusted a pH of 5.1. The resulting cloudiness became pelleted by centrifugation at maximum speed. After that the clear supernatant was added via an FPLC pump a 5 percent. Buffer B portion applied to the column (Order solution thus contains 50 mM NaCl) to get the first foreign pro to elute complexion. The maximum loading was about 8 mg total protein / ml gel. After application, the protein was Gradients up to 0.4 mM NaCl eluted (increase over 22 column vol mina).

The desired gp 250/350 protein eluted between 0.15 and 0.35 mM NaCl portion, with the exact selection continuing on fractions to be purified after polyacrylamide gel electrophoresis (PAGE) was decided. The pillar order was also without Zudo Buffer B possible, but likely as a result of this metering, the gp loading capacity and the Purification factor increased. The low pH in the Chro matography may have been due to similar reasons his. Basically, a separation could also be done with one reach a higher pH value, for example up to pH 7.

If phosphate buffer was used, one could Re-buffering step before the following refurbishment steps to be dispensed with.

III. Ultrafiltration

The selected and pooled protein fractions from step II were by ultrafiltration before application to a gel filter Concentrate the tration column by a factor of about 10 to 20 trated. The final protein concentrations ranged from 1 to 2 mg / ml. Minicon CS15 chambers were used for ultrafiltration (Amicon) used. (For larger solution volumes, ent accordingly larger filtration devices with larger capacity  think, for example, of stirred cells or cross-filtration mo dule.)

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 or Sepharose 6 are considered as BioProcess media.
Buffer system: Isocratic 100 or 200 mM NaCl in a 20 mM phosphate buffer (pH 7).

Flow: 0.2 ml / min corresponds to 2.5 mm / min.

After the column was equilibrated, 150 μl of protein-containing Solution applied, previously concentrated (CS15) and through Centrifugation had been freed from turbid substances. One eluted at a flow of about 2.5 mm / min and separated. United were the elution fractions after PAGE and silver staining gp 250 and or gp 350 would contain no visible impurities ten.

It was found that there was a good separation / purification only with a low protein charge for the proteins (gp). Contrary to the manufacturer's instructions, a load of up to 5 mg was already found with a protein load of 500 µg only a poor separation, while at around 150 ug protein good results have been achieved.

Pure gp 250/350 is the gel filter in pooled gp fractions frozen in stable storage at about -20 ° C. Total was the pure by the purification process about 25% of the product won.  

Comparative Example 1

Example 2 was repeated with the exception that in Spinner reactor did not allow cell confluency and the FCS thinning with cell adherence removed. In doing so were unable to cultivate cells that could be cultivated in protein-free medium will hold.

Claims (6)

1. Hamster 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 ex primed and is obtainable in that one

• the cell line is infected with a vector expressing the glycoproteins (gp) mentioned by transfection,
• the vector comprises a selection marker which is missing from the cell line,
• - The cell line is cultivated and selected with the aid of the selection marker for cells which stably express the glycoproteins after the selection factor (inhibitor) has been omitted.

2. Hamster 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 are expressed in a protein-free medium and can be obtained 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 reactor 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 thereby
• - does not actively remove the adherence of the cells and
• - If necessary, form and detach spheroids from cells, separate the detached cell spheroids, cultivate them in suspension with gentle movement and select 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 hamster cell line according to claim 1 to process steps (A) (a) to (A) (b).

3. hamster cell line according to claim 1 or 2, characterized net that as a hamster cell line a Chinese hamster ovary Cell line (CHO), for example CHO K1 = ATCC CCL61, or one Baby hamster kidney cell line (BHK), for example BHK 21C13 = ATCC CCLIO used. 4. hamster cell line according to any one of the preceding claims, there characterized in that as a selection marker Dihydrofolate reductase gene (DHFR gene) and as an inhibitor Me thotrexat (MTX) used. 5. Hamster cell line CHO K6 = 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) 350, characterized in that

• (a) cultivating a cell line according to one of claims 1 to 5 and allowing the desired glycoproteins to be expressed,
• (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 resulting glycoproteins (gp) are recovered, optionally after fractionation.