WO1987003909A1

WO1987003909A1 - Fermentation processes

Info

Publication number: WO1987003909A1
Application number: PCT/GB1986/000773
Authority: WO
Inventors: Michael Edward Brown
Original assignee: Celltech Ltd
Current assignee: Celltech R&D Ltd
Priority date: 1985-12-17
Filing date: 1986-12-17
Publication date: 1987-07-02
Anticipated expiration: 1988-06-17
Also published as: AU606587B2; AU6776287A; EP0250523A1; GB8531069D0; JPS63503112A

Abstract

A multi-stage, e.g. two stage, continuous culture process, in which host cells transformed with DNA coding for a product are grown in an initial stage in which product formation is uninduced and a subsequent stage in which product formation is induced. Culturing is continued for extended periods of time whilst maintaining satisfactory and sustained levels of product formation by maintaining the specific growth rate of cells in the induced stage at or near the maximum specific growth rate of the cells under the given culture conditions.

Description

FERMENTATION PROCESSES

Field of The Invention

This invention relates to fermentation processes, in particular to multi-stage continuous culture processes in which host cells transformed with DNA coding for a product are cultured and production of the product is induced in a subsequent stage after an inital uninduced stage.

Background to The Invention

Advances in recombinant DNA technology over recent years have made it possible to prepare a wide variety of valuable products by culturing host cells, such as bacteria, which have been transformed with DNA sequences which code for the products. In order to apply this technology industrially, however, high yielding and robust expression systems and processes are required: the products must be produced in sufficient quantity and at a commercially viable cost. In particular, it is a fundemental requirement that the transformed cells can be maintained substantially stably over the large number of cell generations required for large scale industrial production.

Inducible, plasmid-based expression systems have been developed, among other things to overcome problems associated with inherent instability of high copy number plasmid vectors and inhibition of cell growth by foreign product formation. For example, systems such as those described in UK Patent Specification no. 1557755 (Alfred Benzon A/S) and in our copending patent application, GB 2136814A, have been proposed, in which the vector copy number and/or the expression of foreign gene products may be induced by temperature change or other means. This invention relates to culture processes using inducible expression vectors such as those described above, and other expression systems in which production of products coded by heterologous DNA is inducible. Batch and continuous fermentation processes are well known. Continuous processes have economic advantages over batch processes: fermenter capacity is more efficiently used and operation costs are potentially lower in view of the avoidance of the need to harvest and sterilise the fermenter between batches. However, there are inherent problems associated with continuous culture of transformed host cells. Vector-free segregants may arise on host-cell division during growth, and such segregants may have a metabolic advantage over vector-containing cells, especially when expression of the foreign gene product has been induced. After induction, the vector-containing cells are compelled to channel a significant proportion of their metabolic effort into production of a foreign gene product which may have no benefit for the cells. Consequently, following induction, vector-containing cells are not able to grow and divide as rapidly as vector-free cells and vector-containing cells are quickly displaced from the continuous culture.

Similar considerations apply to transformed host cells in which the foreign DNA coding for the product is integrated into the host genome. On culturing such cells, metabolically favoured mutants which have lost the ability to produce the product may arise and displace the productive cells from the continuous culture.

In order to overcome the problem of overgrowth by plasmid-free cells during continuous culture it has been proposed (Siegel R & Ryu D.D.Y., Biotechnology and Bioengineering, Vol. XXVII, pages 28-33 (1985)) to separate the cell growth and product biosynthesis phases of the culture by use of a two stage continous culture system. Siegel & Ryu describe experiments in which E.coli M72 cells containing the temperature inducible plasmid pPL(c)23-trp Al were cultured in a two stage continuous culture system with induction of product formation in the second stage. They observed product formation for a period of about 80 hours, though with significantly decreased rates of product formation with time. Their results did not satisfactorily demonstrate the feasibility of using two stage continuous culture systems industrially for culturing of inducible plasmid containing cells. We have further studied the use of two stage continuous culture systems for culturing inducible plasmid-containing cells. We believe that the lack of success experienced by Siegel and Ryu in achieving a stable state in the second stage was due in part to the inherent instability of the host-vector systems which they used, but more fundamentally because of their apparent lack of appreciation of the need to control the specific growth rate of the plasmid-containing cells in the second stage in order to minimise overgrowth by plasmid-free cells. We have found that by appropriate control of the specific growth rate in the induced second stage it is possible to continuously culture plasmid containing cells for extended periods of time, whilst maintaining satisfactory and sustained levels of product formation.

Summary of The Invention

Accordingly the present invention provides a multi-stage continuous culture process in which host cells transformed with DNA coding for a product are cultured and production of the product is induced in a subsequent stage after an initial uninduced stage, characterised in that, in the subsequent stage the specific growth rate of the transformed cells is maintained at or near to their maximum specific growth rate under the given culture conditions.

Under steady state conditions in a single stage continuous culture the specific growth rate (μ) is equal to the dilution rate

(D); the rate at which cells are washed out of the fermenter is exactly balanced by the growth rate of the cells. The dilution rate

(D) is given by the formula:

where F = the nutrient medium flow-rate (ml/hr), a

V = the culture volume (ml).

In the case of a two stage continuous culture operating under steady state conditions the specific growth rate (μ₁) of cells grown in the first stage equals the dilution rate; whereas specific growth rate (μ₂) in the second stage is given by the formula: D₂ - D₁₂ X₁ X₂

where D₂ = the overall dilution rate for the second stage (hr ^-1); D₁₂ = the partial dilution rate for the second stage determined by the flow rate of culture medium from the first stage to the second stage (hr ^-1), and

X₁ and X₂ = the biomass concentrations in the first and second stages respectively. D₂ is given by the formula:

D₂ = F₀₂ + F₁₂ V₂

where F₀₂ = the flow rate of nutrient medium to the second stage (ml/hr); F₁₂ = the flow rate of culture from the first stage to the second stage (ml/hr), and V₂ = the volume of the culture in the second stage (ml).

Similarly, D₁₂ is given by the formula: D₁₂ = F₁₂

Where F₁₂ and V₂ are as defined above.

A further important feature of a two stage continuous culture is the mean residence time (RT) of cells in the second stage, i.e. the average time every one cell spends in the second stage, determined statistically. The mean residence time (RT) is given by the formula:

RT = 1 D₂

Where D₂ is as defined above. The maximum specific growth rate (μ₂max) of the product producing cells in the second stage at a given mean residence time (RT) may be determined empirically. The specific growth rate (μ₂) of the cells may be varied by adjusting the ratio of the flow rates of culture (F₁₂) and fresh nutrient (F₀₂) whilst maintaining the overall dilution rate (D₂) constant, and the maximum specific growth rate (u max) may be determined; for instance, from the position of the peak of the productivity vs specific growth rate curve.

In the process of the invention the specific growth rate of the transformed cells in second stage may be maintained at or near to their maximum specific growth rate by appropriate choice of flow rate of fresh nutrient medium from the medium reservoir and cell containing culture medium from the initial uninduced stage. Typically the specific growth rate is adjusted so that it is in the range from 65% to 135%, preferably from 75% to 125% and especially from 85% to 115% of the maximum specific growth rate. Most preferably the specific growth rate is maintained at approximately the maximum specific growth rate.

Typically also, the mean residence time of product producing cells in the second stage is sufficient to permit product formation but not so long as to allow undue overgrowth by non-producing cells. We have found that productivity is optimised during the second stage if relatively short mean residence times are used; the lower the mean residence time the higher the productivity provided, of course, the mean residence time is sufficient to permit induction of product formation and accumulation of product. In the case of bacterial host cells transformed with an inducible plasmid we have found that mean residence times of less than 4 hours preferably less than 3 hours and especially about 2 hours or less give advantageous productivity levels from the second stage.

Clearly the requisite mean residence time for optimisation of productivity will depend upon the particular host cell and expression system used. Factors such as the rate at which the cells are able to grow and the time required for induction of product formation must be taken into account. Generally, however, it appears that mean residence times of the same order as the cell generation period i.e. the period between cell divisions, give optimum productivities. For instance, mean residence times in the range from one half to two times, preferably from one half to one and a half times the cell generation period may be used.

We have further found that the inherent stability of the uninduced transformed cells, e.g. host-vector system, is an important, though not essential, contributory factor for achieving a stable state in the second stage. If the plasmid is not stable in the first stage, a mixed population of producer and non-producer cells will seed the second stage of the culture. Since non-producing cells are able to grow at faster rates than induced producing cells, the non-producing cells soon overgrown the producing cells with the result that product formation ceases as the producing cells are washed out of the fermenter. It is thus desirable to use expression systems, e.g. host vector systems, of high inherent stability for multi-stage continuous culture. Preferred bacterial host vector systems include the dual origin vector systems described in our copending patent application, GB2136814A.

Even with very stable host-vector systems, overgrowth by vector-free cells will eventually occur because if even a very low level of vector-free cells arises during the initial uninduced stage, after induction of product formation in the subsequent stage vector-free cells can grow at faster rates than vector containing cells. However, in the process of the invention overgrowth is controlled by appropriate adjustment of mean residence time and specific growth rate and advantageously occurs only slowly and over a considerably extended period of time.

The process of the invention is generally applicable to multi-stage continuous culture of transformed host cells in which production of a heterologous product is induced in the second and subsequent stages of the culture. The process is particularly applicable to two stage continuous culture. Any suitable inducible host cell expression system may be used including bacterial, yeast and higher eucaryotic e.g. mammalian, host cell expression systems.

Using the process of the invention we have found that it is possible to maintain product formation in two stage continuous culture for periods of up to several weeks by adjustment of mean residence time and specific growth rate of plasmid containing cells in the second stage. The level of plasmid free cells present in the culture is kept low for an extended period of time and productivity is optimised by maintaining cells for a relatively short mean residence time in the second stage and by causing plasmid containing cells to grow at specific growth rates in the region of their maximum specific growth rate.

Brief Description of Drawings

The invention is described by way of illustration only in the following example which refer to the accompanying diagrams, in which:

Figure 1 - is a schematic representation of a two stage continuous culture system;

Figure 2 - is a photograph of a PAGE gel showing level of product formation at various times after induction in the second stage of a two-stage continuous culture operated at a 2 hour mean residence time;

Figure 3 - is a graph showing accumulated results of the variation of productivity against specific growth rate for 2(O), 3 (●) and 4 (X) hour mean residence times during the second stage of a two stage contnnuous culture, and Figure 4 - is a similar graph showing variation of plasmid free cell population in the second stage as compared with the first stage against specific growth rate for

2(O), 3 (●) and 4 (X) hour mean residence times.

EXAMPLE

MATERIALS AND METHODS

Bacterial Strain & Plasmid

E.coli E103S met+ (Celltech code 1B373) was used as the host strain for the dual origin plasmid ρMG169. The plasmid ρMG169, described in UK Patent-Application No. 2136814A contains the gene coding for Chloramphenicol acetyltransferase under transcriptional control of the tryptophan promotor.

Inoculum Preparation

A single colony isolate was picked from an L agar plate supplemented with 100μg/ml carbenicillin, inoculated into 50 mls of

L Broth in a baffled 250 ml flask and shaken on an orbital shaker at 30ºC. When sufficient growth had occurred this culture was used to inoculate the two chemostat fermenters under aseptic conditions.

Chemostat Medium

The medium used was a carbon limited defined chemostat medium containing glucose as the sole, carbon and energy source as described by Evans et. al. Methods in Microbiology Vol II 1970 and the antifoam polypropylene glycol 2025 was added as required.

The Two Stage Chemostat

With reference to Figure 1, the two stage chemostat used consisted of two mechanically stirred jar fermenters each of total volume 2.5 litres (supplied by MBR Bioreactors A.G. of Switzerland): fermenter 1, the growth fermenter in which uninduced cells were grown, and fermenter 2, the production fermenter in which product formation was induced. Fermenters 1 and 2 were fed with fresh nutrient medium via feed lines 4 and 5 by adjustable flow rate pumps P1 and P2 (Gibson Minipuls peristatic pumps). Cell-containing culture medium was fed from Fermenter 1 to Fermenter 2 via feed line 7 by a similar adjustable flow rate pump P3. Product was withdrawn from fermenter 2 through product line 9. In use the flow rates of pumps P1 (F₀₁), P₂ (F₀₂) and p₃ (F₁₂) are adjυsted as required and the volumes of culture in fermenter 1 (V₁ = 1600ml) and fermenter 2 (V₂ = 1040ml) were set at constant levels by means of overflow pipes (not shown).

The temperature of the culture in the fermenters was controlled in use to repress or induce product synthesis. The pH of the cultures was controlled at ρH7.0 in both fermenters by addition of sterile 1M H₂SO₄ and 3M NaOH through peristatic pumps (not shown). The dissolved oxygen tension was controlled by a combination of stirrer speed and sparged air flow, to ensure that a value in excess of 30% saturation was maintained.

Continuous Culture Experiments - General

The two fermenters were sterilised and filled with medium. The fermenter temperatures were set to 34ºC to ensure that product synthesis remained repressed, after the medium had been inoculated with inoculum prepared as described above. The pumps were started after exponential growth had ceased due to glucose substrate being depleted from the medium. To ensure a steady state existed, three volume 'replacements' were permitted to pass through each vessel before sampling. The temperature in the second vessel was then raised to 38ºC thereby switching on product synthesis. Three volume 'replacements' were permitted to pass through the second stage before sampling.

Sampling was performed under aseptic conditions to provide material for determination of cell and substrate concentrations, plasmid stability determinations and the determination of intracellular product concentration.

Cell concentration was measured by optical density at 600 nm using a PYE Unicam PU 8610 spectrophometer.

The proportion of cells carrying the plasmid was determined by diluting the culture and plating onto L agar at 30ºC. One hundred single colonies were 'picked' onto L agar containing carbenicillin and onto L agar used as a control. The number of colonies resistant to the antibiotic was expressed as a percentage of the number growing on the L agar. Alternatively, samples were diluted and plated onto L agar at 30ºC and 42ºC. Colonies capable of growth at 42ºC did not bear the plasmid and therefore did not induce product formation. The viable count at 42ºC was expressed as a percentage of the viable count at 30ºC and this was also taken to represent the proportion of the population which carried the plasmid.

Product concentration was determined by gradient PAGE and enzyme activity analysis.

Continuous Culture Experiment A - Mean Residence Time (RT) = 2 hr; specific growth rate (μ₂) = 0.25 hr

The two stage chemostat was set up such that in the second stage (fermenter 2) the mean residence time of cells, was 2 hours and the specific growth rate of the cells was approximately 0.25 hr The flow rate of nutrient medium to the first stage was set at 400 ml/hr and to the second stage at 260 ml/hr, and cell-containing culture was pumped from the first stage to the second stage also at 260 ml/hr. The experiment was continued for a period in excess of 284 hr (92 generations) following induction of product fermentation.

After 284 hr the proportion of plasmid-free cells in the first stage fermenter rose from 0.04% at the beginning of the experiment to 0.38%; whereas over the same period the proportion of plasmid-free cells in the second stage fermenter rose to 2.5% of the total cell population.

Product accumulation and increase in the copy number of the plasmid were observed in the second stage fermenter within 6 hr after the temperture of the culture in the second stage had been raised to 38ºC i.e. after induction. With reference to Figure 2, a near constant specific rate of chloramphenicol acetyl transferase

(CAT) synthesis was observed over a period in excess of 168 hr.

Figure 2 is a photograph of a polyacrylamide gel showing the protein/product profile in samples of culture from the second stage fermenter over a period of 168 hr following induction. The gel shows a substantially constant and sustained large band corresponding to the CAT product. A mean value of specific rate of CAT synthesis (Q CAT) of 20.06 mg/g (dry cell weight/hr) was observed.

Continuous Culture Experiment B - Determination of μ_2max at a mean residence time of 2 hr

A further experiment was carried out in which, whilst maintaining the mean residence time in the second stage at 2 hr. the specific growth rate was adjusted to, and maintained at, various values between 0.1 and 0.3 hr. The culture from the second stage was sampled and analysed for each specific growth rate. The maximum specific growth rate at a mean residence time of 2 hr was determined to be approximately 0.23 hr^-1 from the results obtained. The maximum specific growth rate is given by the position of the peak in the curve of specific rate of CAT synthesis (productivity) against specific growth rate - see Figure 3.

Continuous Culture Experiment C - Determination of μ_2max at a 3 hour Mean Residence Time

Similar experiments were carried out in which the mean residence time of cells in the second stage was held constant at 3 hr and the specific growth rate was adjusted to, and maintained at, various values between 0 and 0.25 hr^-1

Continuous Culture Experiment D - Determination of μ_2max at a 4 Hour Mean Residence Time

Similar experiments were carried out in which the mean residence time of cells in the second stage was held constant at 4 hours and the specific growth rate was adjusted to and maintained at, various values between 0 and 0.2 hr^-1

The accumulated results of continuous Culture Experiments A, B and C and given in Figures 3 and 4.

Figure 3 shows curves of productivity (specific rate of CAT synthesis - Q CAT) during the second stage against specific growth rate for the three choosen mean residence times: 2 hr (O), 3 hr (O) and 4 hr (X). The maximum specific growth rate decreases with increasing mean residence time.

Thus when RT = 2 hr μ_2max = approximately 0.23^-1 when RT = 3 hr μ_2max = approximately 0.15^-1 when RT = 4 hr μ_2max = approximately 0.12^-1 Also productivity decreased with increasing mean residence time; productivity (specific rate of CAT synthesis - Q CAT) was highest at the 2 hr mean residence time and lowest at the 4 hr mean residence time.

Figure 4 is a graph showing curves of the proportional increase in the plasmid-free cells in the second stage compared with the equivalent time point in the first stage against specific growth rate in the second stage, for the three chosen mean residence times, 2 hr (O), 3 hr (●) and 4 hr (X). The sharp increase in the plasmid-free population corresponds to specific growth rates in the region at or above the maximum specific growth rate. Thus this increase in plasmid free cells occurs at the highest specific growth rate when RT = 2 hr and at the lowest specific growth rate when RT = 4 hr.

1. A multi-stage continuous culture process in which host cells transformed with DNA coding for a product are cultured and production of the product is induced in a subsequent stage after an initial uninduced stage, characterised in that, in the subsequent stage the specific growth rate of the transformed cells is maintained at or near to their maximum specific growth rate under the given culture conditions.

2. A process according to Claim 1, in which the specific growth rate of the transformed cells in the subsequent stage is in the range from 65% to 135% of the maximum specific growth rate.

3. A process according to Claim 1, in which the specific growth rate of the transformed cells in the subsequent stage is maintained at approximately the maximum specific growth rate.

4. A process according to Claim 1, in which the mean residence time of cells in the subsequent induced stage is from one half to two times the cell generation period.

Claims

5. A process according to Claim 1, in which the transformed cells are bacterial host cells transformed with an inducible plasmid expression vector.

6. A process according to Claim 5, in which the mean residence time of cells in the subsequent induced stage is less than 4 hours.

7. A proccess according to Claim 6, in which the mean residence time of cells in the subsequent induced stage is 2 hours or less.

8. A process according to any one of Claims 1 to 7, which is a two stage continuous culture process.