GB2085894A - Process for the production of interferon - Google Patents

Abstract

A process for the production of interferon from white blood or lymphoblastoid type cells in which the cells are cultured at a conventional temperature, induced by addition of a virus and incubated with the virus, characterised in that for a period of time, before, during or after induction, effective to prolong the half-life of interferon mRNA produced in the cell after induction, the temperature is reduced. The improved process is of particular value in producing human interferon from human peripheral blood lymphocyte or lymphoblastoid cells.

Description

SPECIFICATION Process for the production of interferon This invention relates to the production of interferon.

In conventional processes for the production of interferon in white blood cells or lymphoblastoid cells by induction with a virus, the cells are cultured at a temperature of about 37"C, induced at this temperature by the addition of a virus, thereby initiating the production of interferon and incubated with the virus while maintaining the temperature in the region of 37"C.

It has now been found that significantly higher yields of interferon can be produced by reducing the temperature of the cultivated cells. The temperature can be reduced before, during or after the induction with a virus. The effect of reducing the temperature is to alter the balance of competitive processes within the cell. Induction of the cell with virus produces mRNA coding for interferon. Some of the mRNA produced is retained on the polysomes, where some of it is translated into interferon. Untranslated and other surplus mRNA becomes degraded. The general slowing down of cell processes caused by lowering the temperature does not affect all processes occuring within the cell to the same extent.It has been found experimentally that it increases the half-life of mRNA produced in the cell, with the result that the mRNA is translated into interferon over a longer period of time than at the conventional temperature. Although the rate of production of interferon is lower, the total yield is increased. This improvement in total yield is obtained when the temperature is reduced at any time typically from about 40 hours before induction, to a short period before the production of interferon at the conventional temperature is complete. It is normally complete at about 9-1 2 hours after induction, at the conventional temperature.

It is known that a temperature reduction enhances the yield of interferon produced by diploid fibroblast cells or rabbit RK13 cells which are very similar to diploid fibroblasts. However, fibroblast cells behave very differently from white blood cells and lymphoblastoids in an interferon-producing context. Salient and fundamental differences include the following:1. Fibroblasts synthesise mainly P-IFN; white blood and lymphoblastoid cells mainly a.

2. Fibroblasts are adherent to a solid surface, whereas white blood and lymphoblastoid cells are not.

3. Fibroblasts, but not white blood or lymphoblastoid cells, can be induced with doublestranded RNA.

4. Fibroblasts, but not white blood or lymphoblastoid cells, can be superinduced. (Superinduction involves inhibiting translation of interferon from its mRNA temporarily, after sufficient mRNA has been formed preventing transcription of further mRNA from the gene and then reversing the first inhibition to allow translation of interferon from mRNA to proceed.) 5. Fibroblasts do not give more interferon when pretreated with butyrate or bromodeoxyuridine, whereas white blood cells and lymphoblastoid cells do.

From this list, which could be extended, it is clear that treatments that affect yields in fibroblasts do not do so in leukocytes and lymphoblastoid cells. Therefore the fact that the temperature drop works in white blood cells and lymphoblastoid cells was not predictable from the fact that it was shown to work in cells of the fibroblast type.

It is also known that certain virus inducers are temperature-sensitive, requiring a low temperature (below 35"C) to synthesise viral RNA or protein. Examples are certain mutants of Semliki Forest Virus. The present invention is not concerned with use of these unusual types of virus as inducers.

According to the present invention there is provided a process for the production of interferon from white blood or lymphoblastoid cells or similar cells also having the five characteristics defined above, in which process, cells are cultured at a conventional temperature, usually from 35 to 38"C, especially about 37"C, induced by addition of a virus and incubated with the virus, but excluding a virus which requires a temperature below 35"C in order to synthesise viral RNA or protein, characterised in that for a period of time, before, during or after induction, effective to prolong the half-life of interferon mRNA produced in the cell after induction, the temperature is reduced, preferably to from 25 to 33"C, especially to about 28"C.

The cells are most conveniently cultured at about 37"C. At too low a temperature, growth is too slow while at too high a temperature the cells are destroyed by heat. Ordinarily a temperature of from 35 to 38"C is the most suitable range.

The temperature of the cell medium is shifted, i.e. reduced, from that at which the cells were cultured. If the shift is to too low a temperature no improvement in yield is observed, while a very small shift does not produce the maximum improvement in yield.

Laboratory experiments have shown that the yield of interferon plotted ageinst the reduced temperature chosen rises steeply from about 21 or 22"C, reaching a peak typically at about 28"C, and declines thereafter, but less steeply than at the low temperature side of the peak.

Generally, a reduction in temperature of from 5 to 1 5,C lower, preferably 8 to 1 0 C lower, than the culturing temperature can be expected to give an importantly improved yield.

The timing of the temperature reduction must be chosen sensibly if significantly higher yields of interferon are to be obtained. Sufficient time must be allowed for the interior of the cells to acquire the reduced temperature of the medium in which they are present. The objective is that the cell process giving rise to a longer half-life or residence time of interferon mRNA within the cell should become preferred to competitive cell processes. The residence time of interferon mRNA in the cell can be measured experimentally by extracting total RNA from the cells at intervals, microinjecting it into oocytes (a technique known in itself) and assaying the interferon produced by the oocytes. It is preferable, for convenience, to extract total RNA produced by the whole cell, but an alternative way of monitoring the mRNA is to extract only RNA associated with the polysomes.

The advantages of the invention are believed to be secured most easily by reducing the temperature and maintaining a reduced temperature for the remainder of the process. For example, if the temperature is reduced before induction it is preferably maintained at this or some other reduced level up to and through induction and incubation, until the process is stopped. Similarly if it is reduced after induction, a reduced temperature is preferably maintained from then onwards during the incubation process. While some variation of the level of the reduced temperature is permissible, it is ordinarily desirable to maintain a substantially constant level of reduced temperature during the remainder of the process. Such a procedure will hereinafter be referred to as the reduced constant temperature procedure.

In the reduced constant temperature procedure, the temperature is preferably shifted between 40 hours before and at least 9 or 10 hours after induction. If the production of interferon does not continue for as long as 9 or 10 hours, the process could, of course, be stopped earlier.

Laboratory experiments have indicated that the best yields are obtained when the shift takes place from 10 to 20 hours before induction or 3 to 10 hours after induction. An advantage of shifting before induction is that the reduced temperature saves some heating costs. A shift after induction is preferred at from 3 to 7 hours thereafter. The experimental evidence is that a shift at around 7 hours post-induction gives maximum yield, although the exact optimum timing of the shift to optimise the yield of interferon will probably depend on the scale of the process operated and/or the concentration of cells in the medium. A shift substantially simultaneous with induction including up to 1 hour thereafter, is not particularly preferred.It is expected that there will be a balance of process economics between, on the one hand, optimising the yield of interferon by shifting after induction and, on the other hand, optimising reduction of heat costs by shifting before induction.

Particularly marked effects have been shown with one of the important interferon-producing techniques in current use, i.e. Sendai virus induced production in Namalwa cells, but the effect is not limited to a particular kind of cell or virus inducer, except as hereinbefore mentioned. For example, the method can also be used with human leukocytes, especially human peripheral lymphocytes, obtained from fresh human blood. Human leukocytes and other cells to which the precess may be applied are described by Cantell in ''Interferon 1, 1 979" Academic Press, New York, 1979 page 1, by Kauppinen, Myllylla and Cantell in "Human Interferon" edited by Stinebring and Chapple, page 1, Plenum Publishing Corporation, and by W.E. Stewart II in "The Interferon System" Springer Verlag 1979.Normal Sendai, defective Sendai and mixtures of these types of virus have been used successfully in the process of the invention. Other viruses, for example Newcastle Disease Virus, could be used.

Preferably the cells are treated before induction with a chemical enhancer of the yield of interferon, e.g. butyric acid or 5-bromodeoxyuridine.

The interferon produced by the process of the invention is believed not to be significantly different from that produced by the conventional (37"C throughout) process. In the conventional process Namalwa cells produce a mixture of alpha- and beta- interferons. One of the characteristics of alpha-interferon is that the ratio of its titre on EBTr cells to its titre on HFF cells is high, whereas the converse is true for beta-interferon. No difference in the ratios of activity on EBTr cells to HFF cells was observed between the conventional process and the process of the invention. This result was confirmed (i) by titration against anti-alpha interferon and anti-beta interferon sera, (ii) by affinity for a monoclonal antibody to alpha-interferon and (iii) by radio immuno-assay using the monoclonal antibody to alpha-interferon.

The process of the invention will ordinarily include the further step of extracting the expressed interferon from the cells or separating it from cell solids and separating it also from the incubation medium. Any procedure known per se for separating interferon and working it up can be used. The invention includes the interferon thus produced, which may, if desired, be purified against one or more appropriate antibodies, e.g. by affinity chromatography on monoclonal or other bound antibodies to interferons.

The following Examples illustrate the invention. Examples 1-6 and 8 relate to temperature shifts at or after induction, Example 7 to temperature shifts before, during and after induction.

In Examples 1-7 Namalwa cells are used and in Example 8 leukocyte (human peripheral blood lymphocyte) cells. In all cases an improvement in yield resulting from the temperature shift was observed. Example 6 correlates this improvement in yield with increased half-life of cellular interferon mRNA.

The Examples refer to the accompanying drawings, in which: Figure 1 is a graph of cumulative yield of interferon at 24 hours after induction on the y-axis (ordinate), against the reduced temperature selected for the shift, on the x-axis (abscissa); Figure 2 is a graph of cumulative yield of interferon at 24 hours after induction on the y-axis, against the time at which the temperature was shifted after induction, on the x-axis; Figure 3 is a graph of cumulative yieid of interferon, on the y-axis, against the time after induction at which the yield was measured, on the x-axis, for three experiments, two (2, 3) with temperature shift and one without (1); Figure 4a is a graph of cumulative yield of interferon on the y-axis against the time after induction at which the yield was measured, on the x-axis, similar to plots 1 and 3 of Fig. 3;; Figure 4b is a graph of yield of interferon obtained by injecting RNA extracted from cells into oocytes, on the y-axis, against time of extraction on the x-axis corresponding to the times of Fig.

4a; and Figure 5 is a graph of similar kind to Fig. 2 except that it includes shifts before induction.

Methods used in Examples 1-6 (a) Cells and Virus Namalwa cells obtained from the Wellcome Research Laboratories, Beckenham, Kent, were grown in RPM1 1640 buffered with 20 mM Hepes containing 10% newborn calf serum, (NCS, Flow Laboratories, Irvine, Scotland), 60 micrograms/ml penicillin and 100 micrograms/ml of streptomycin.

When a cell count of 1.5 X 106 to 2 > c X 106/ml was reached, the cells were diluted in the same medium to a concentration of 0.5 > < X 106/ml. EBTr cells, a gift from Dr. J. Vilcek, were grown in Glasgow Modified Eagles Medium (GMEM) containing 10% NCS. Sendai virus was grown in the allantoic cavity of 1 0-day-old eggs and was harvested after 72 hours. The resultant virus was a mixture of defective and normal types. Each batch of virus was tested by titration for the optimal dose for interferon induction.

(b) Pre-treatment of cells In Examples 1-4 and 6 the cells were treated for 48 hours with butyric acid before induction.

(c) The process Namalwa cells were centrifuged (2000 g for 5 minutes), resuspended in RPM1 1640 with 2% NCS at 1 X 106 cells/ml, and incubated at 37"C for 48 hours with 1 mM butyric acid.

After centrifugation the cells were resuspended at 2 x 106/ml, Sendai vdirus added, and incubated for a further period in temperature-controlled water baths. Subsequently, the cells were centrifuged and the supernatant was dialysed against 50 volumes of pH2 buffer overnight and then against PBS for 8 hours before it was assayed for interferon.

When the titre of intracellular interferon was to be measured, the cells were washed three times in 1 ml of cold PBS, lysed by addition of 30 microlitres of concentrated HCI, diluted to 1 ml with RPM1 1640 containing 2% NCS, and the cell debris was removed by centrifugation before the samples were dialysed.

(d) Interferon assay Interferon was assayed by the RNA-reduction method (Atherton 8 Burke, 1975) using EBTr cells. The assay was performed 24 hours after induction except where otherwise stated. The results obtained in the assay were converted into international units, based on the human leukocyte interferon research standard (69/19). The conversion factor used was: 1 unit assayed using EBTr cells = 0.1 I.U.

Example 1 The optimum temperature of incubation was determined by inducing the cells at 37"C and subsequently shifting cells from 37"C to another temperature at 7 hours after induction and assaying the interferon produced after 24 hours. The results are shown in Fig. 1. The largest enhancement was observed when the cells were shifted to 28"C, but only at 19"C were yields lower than at 37"C incubation throughout. Raising the temperature of incubation to 41 C inhibited interferon production.

Example 2 Example 1 was repeated except that the temperature was reduced 3Q hours after induction.

The yields showed a similar temperature dependence to that of Fig. 1 and were higher than for incubation at 37"C throughout. However, they were lower than in Example 1.

Example 3 The effect of the length of incubation at 37"C on the enhancement of interferon yield was investigated by inducing the cells at 37"C and shifting the temperature to 28"C at various times after induction. The results are shown in Fig. 2. The optimum time to shift the temperature was 7 hours after induction, but an enhancement could be seen at all times up to 10 hours. The higher yield caused by changing to 28"C at induction, rather than at 1 hour post-induction is reproducible, but its cause is unknown. In Namalwa cells at 37"C interferon production has usually ceased by about 10 hours after induction. Thus it appears that the shift to a lower temperature can enhance yields at any time up to the cessation of interferon synthesis.

Example 4 Three experiments were performed in which interferon was assayed at various times after induction at 37"C and in which (1) the temperature was 37"C throughout, (2) the temperature was shifted to 28"C at 32 hours after induction and (3) the temperature was shifted to 28"C at 7 hours after induction. The results are shown in Fig. 3. At the lower temperature, interferon accumulation is slower but continues for longer.

In this experiment intracellular titres were also measured and found to be higher in the cells incubated at lower temperature. Therefore the enhanced yields at the low temperature cannot be accounted for by increased secretion, but are accounted for by prolonging the period of interferon synthesis.

Example 5 In the Examples 1-4 the cells were treated for 48 hours with butyric acid before induction.

The enhancement of yields was also demonstrated in untreated cells as well as in cells treated with 5-bromodeoxyuridine for 48 hours before induction.

The proportionate enhancement in yield obtained by lowering the temperature at 3 2 hours or 7 hours after induction was approximately the same, although overall yields were different. Thus pre-treatment of the cells is not the major factor giving the enhancement of yield.

Example 6 Experiments were carried out in which Namalwa cells were grown at 37"C, treated with butyric acid and induced with the Sendai virus and the temperature was reduced 7 hours later.

Cumulative yields of interferon obtained from the cells after various times are plotted in Fig. 4a.

Interferon was produced more slowly by the cells incubated at 28"C but the cumulative yield was greater.

Amounts of interferon mRNA present in the induced cells at various times were measured by extracting RNA, microinjecting it into oocytes and subsequently assaying for the interferon produced. The amounts of interferon mRNA at a time soon after the temperature was reduced were similar at the two temperatures but at later times more interferon mRNA was present in cells maintained at the lower temperature (Fig. 4b). These results support the view that the prolonged synthesis of interferon at 28"C and concomitant improvement in yield, are due to a lengthening of the half-life of interferon mRNA in the cell.

In these experiments interferon synthesis at 37"C ceased at times between 9 and 12 hours after induction. However, in all cases interferon synthesis continued for longer at 28"C, halting between 1 5 and 22 hours after induction. In all experiments interferon mRNA was present for longer at 28"C than at 37"C. The loss of interferon mRNA activity at 37"C was not due to cell death because even at 24 hours after induction all the cells at either temperature still excluded trypan blue.

Since mRNA can be present in the cells but not translated, it was necessary to look at the levels of interferon mRNA on the polysom s. Cells were induced and the temperature was reduced to 28"C at 7 hours after induction and the interferon yields were followed. The results were similar to those shown in Figs. 3 and 4a. Polysomes were also prepared from these cells, and the RNA was subsequently extracted from them and assayed by microinjecting into oocytes.

The results showed that there was more interferon mRNA present on the polysomes at later times at the lower temperature (see the "Table" below). Therefore, the enhanced yields of interferon at 28"C were due to the prolonged presence of more interferon mRNA on the polysomes. At 375C interferon mRNA was still present on the polysomes at times after induction when interferon synthesis had apparently halted.

TABLE Time of measurement of mRNA on polysomes, process temperature Interferon Yield conditions (units/ml) 14 hours post-induction, 37"C throughout 40 14 hours post-induction, shift to 28"C at 7 hours post-induction 1 25 16 hours post-induction, 37"C throughout 1 6 1 6 hours post-induction, shift to 28"C at 7 hours post-induction 25 Example 7 Namalwa cells as in Examples 1-6 were suspended at 1 X 106 cells/ml in RPM 1 1 640 + 2% calf serum in some cases containing 1 mm butyric acid.The cells were incubated at 37"C for 48 hours, pelletted by centrifugation and resuspended at 2 X 1 06/my in fresh medium containing Sendai virus as in Examples 1 - 6. As before, the optimum amount of Sendai virus was determined by titration. The temperature was shifted from 37"C to 28"C at various times before induction, simultaneously with induction and after induction. After 24 hours cells were pelletted and the interferon present in the medium was measured 24 hours after induction. The results, shown in Fig. 5, demonstrate an improvement in yield at various times within the range 8 to 40 hours before induction, with and without the addition of butyric acid. A better improvement of yield is obtained when the shift is made 7 hours after induction.

Example 8 In this experiment human blood was used. Lymphocytes were prepared from Buffy coats of the blood using Ficoll-Paque. They were resuspended in RPM1 1640 + 10% foetal calf serum and incubated overnight at 37"C. After centrifuging and resuspending the cells, Sendai virus was added and they were incubated at 37"C for 24 hours. Some cultures were incubated at 28"C from 5 hours after induction. Interferon was then measured in the medium at 24 hours after induction. The yields of interferon were 37"C throughout 3.8 log10 unit/ml 28"C from 5 hours post-induction 4.6 log1O unit/ml

Claims (11)

1. A process for the production of interferon from white blood or lymphoblastoid type cells in which the cells are cultured at a conventional temperature, induced by addition of a virus and incubated with the virus, but excluding a virus which requires a temperature below 35"C in order to synthesise viral RNA or protein, characterised in that for a period of time, before, during of after induction, effective to prolong the half-life of interferon mRNA produced in the cell after induction, the temperature is reduced.

2. A process according to Claim 1 characterised in that the temperature is reduced by 5 to 15"C lower than the culture temperature.

3. A process according to Claim 2 characterised in that the temperature is reduced by 8 to 10"C lower than the culture temperature.

4. A process according to Claim 1 characterised in that the culture temperature is from 35 to 38"C and the temperature is reduced to one within the range 25 to 33"C.

5. A process according to Claim 1, 2, 3 or 4 characterised in that the temperature is reduced 10 to 20 hours before induction and the cells are kept at substantially the same reduced temperature until at least 9 hours after induction.

6. A process according to Claim 1, 2, 3 or 4 characterised in that the temperature is reduced 3 to 7 hours after induction and the cells are kept at substantially the same reduced temperature until at least 9 hours after induction.

7. A process according to any preceding claim characterised in that before induction the cells are treated with a chemical enhancer of the yield of interferon.

8. A process according to Claim 7 characterised in that the enhancer is butyric acid or 5bromodeoxyuridine.

9. A process according to any preceding claim characterised in that human peripheral blood lymphocyte or human lymphoblastoid cells are used as the starting cells.

1 0. A process according to any preceding claim characterised in that the interferon produced is extracted from the cells or separated from cell solids and is separated from the incubation medium.

11. A process according to Claim 10 characterised in that the interferon produced is purified against an antibody or antibodies thereto.

1 2. A process according to Claim 1 substantially as hereinbefore described with reference to any one of the Examples.

1 3. Interferon produced by a process claimed in any preceding claim.