Patent Application: US-14277898-A

Abstract:
a process of culturing a microorganism in a culture medium in which process the addition of feed medium is controlled by using the production of a by - product as a measure of the culture conditions , characterized in that the by - product is an electrically charged metabolite produced by the microorganism , and in that the production of the metabolite is monitored by measuring the conductance of the culture medium . the metabolite may be acetate and the microorganism may be yeast which is genetically engineered to produce a desired polypeptide .

Description:
preferred aspects of the invention will now be described by way of example and with reference to the accompanying drawings in which : fig1 is a representation of some key parameters during a fed - batch fermentation of a yeast strain producing recombinant human albumin . the points at which the feed addition was started and finished are indicated by arrows . fig2 shows parameters for part of a fed - batch fermentation during which , at the indicated time , a deliberate , sudden 20 % feed rate increase was applied . fig3 is a similar experiment as shown in fig2 ; however , in this case a 40 % step increase was applied to the feed addition rate . fig4 is a simplified flow chart of a typical feed rate control algorithm , using the electrical conductance signal , that was used in the experiment represented in fig5 . fig5 is the representation of some parameters in an experiment during which the exponential factor k was set at 0 . 12 h - 1 which is higher than the usual value of 0 . 07 h - 1 for this yeast strain . during the experiment the algorithm using the conductance signal , of which the flow chart is shown in fig4 was active . fig6 shows some parameters of an experiment with the bacterial strain e . coli dh5α in which the conductance control algorithm shown in fig4 was active . the factor k was set at 0 . 4 h - 1 in this experiment . normally a factor 0 . 11 h - 1 would be used ( riesenberg et al ., 1991 ). fig7 shows some parameters of an experiment with the bacterial strain e . coli dh5α in which the feed rate was manually increased in three steps ( 21 . 3 - 22 . 3 h ) and then was controlled by a similar algorithm as described in fig4 but modified as described in example 5 . fig8 is a schematic representation of a fermenter suitable for use in the process of the invention . in order to determine the normal trend of the electrical conductance during a fed - batch fermentation ( fig1 ), we monitored the conductance on a fermentation control computer linked to an aber instruments ( aberystwyth , uk ) biomass monitor 214a with an aber instruments capacitance probe . the conductance signal was noisy due to the aeration of the fermenter . therefore , the conductance had to be electrically filtered using the supplied filter number 2 on the biomass monitor 214a . as an alternative other conductivity probes and monitors can be used as long as the signal is adequately filtered to smooth the noisy signal . one such set up can be a broadley james conductivity probe ( from ft applikon ) linked to an mcd43 monitor ( lth electronics ) which uses a 3 min filter . all data in fig1 are averaged over 10 min ( due to the data storage limitations of the fermentation control computer ). the fermentation was performed as described by clarke et al ( 1990 ), which is incorporated by reference . essentially , the fermentation was as follows . the fermentation was based on yeast transformed to express recombinant human albumin ( rha ). the cloning strategy for construction of the yeast was as disclosed in ep 431 880 . a stock master cell culture in defined liquid medium ( buffered minimal medium ( bmm ) salts medium : yeast nitrogen base [ without amino acids and ( nh 4 ) 2 so 4 , difco ], 1 . 7 g / l ; citric acid monohydrate 6 . 09 g / l ; anhydrous na 2 hpo 4 , 20 . 16 g / l ; ph 6 . 5 ± 0 . 2 ; ( nh 4 ) 2 so 4 , 5 g / l ; sucrose is added to 20 g / l ) was used to prepare running stocks ( manufacturer &# 39 ; s working cell bank ) of process yeast suitable for the preparation of shake flask cultures by freezing aliquots of the culture in the presence of 20 % ( w / v ) trehalose . shake flask culture . the yeast [ cir °, paye316 ] was grown as an axenic culture physiologically suited for inoculation of the seed vessel . if timing of the seed vessel is to be reproducible , it is necessary to define the phase of growth ( primary carbohydrate excess ) and inoculum biomass ( 12 ± 2 mg / l which requires a 100 ml inoculum per 10 liters of medium ). one stock vial was inoculated into a shake flask containing 100 ml of bmm + 2 % ( w / v ) sucrose and the flask was incubated at 30 ° c . on an orbital shaker ( 200 rpm revolutions per minutes ) until a cell dry weight ( cdw ) of 0 . 6 - 1 . 2 g / l ( assessed by optical density at 600 nm ) was obtained . this culture was then used to inoculate a seed fermentation vessel to a level of 12 ± 2 mg / l . seed fermentation . the inoculum for the main production fermenter was provided by growing the production organism , preferably s . cerevisiae [ cir °, paye316 ], in a seed fermenter to a high cell dry weight of approx . 100 g / l . a fed - batch regime was followed so as to minimise the accumulation of ethanol and acetate and thus to maximise cell yield . the whole of each fermentation was monitored and controlled via a computer control system , such as the multi - fermenter computer system ( mfcs ) software available from b . braun ( germany ). the software supplied by b . braun is a supervisory control and data acquisition package ; similar packages are available from other companies . the algorithm is intended to control the addition of sucrose so that maximum biomass is achieved by avoiding the crabtree effect , thereby minimising the production of ethanol and / or acetate . the fermentation vessel was subjected to a hot naoh wash and pyrogen - free water ( pfw ) rinse . the heat sterilised vessel contained one volume of sterile mw10 nedium ( table 1 ) batch salts plus trace elements . an alternative medium is given in table 2 . clearly , the initial conductivity will vary according to the constitution of the medium . the medium for rha production can be ultrafiltered ( 10 , 000 mol . wt . cut - off ) to remove endotoxins . table 1______________________________________mw10 medium______________________________________constituents batch medium feed medium______________________________________ saltskh . sub . 2 po . sub . 4 2 . 74 g / l 10 . 9 g / l mgso . sub . 4 . 7h . sub . 2 o 0 . 58 g / l 2 . 3 g / l cacl . sub . 2 . 2h . sub . 2 o 0 . 06 g / l 0 . 24 g / l h . sub . 3 po . sub . 4 ( 85 % w / w ) 0 . 88 ml / l 1 . 76 ml / l vitamins ca pantothenate 20 mg / l 180 mg / l nicotinic acid 33 . 3 mg / l 300 mg / l m - inositol 20 mg / l 180 mg / l d - biotin 0 . 133 mg / l 0 . 8 mg / l thiamine . hcl 16 mg / l 32 mg / l trace element stock 10 ml / l 20 ml / l sucrose 0 * 500 g / l______________________________________ trace element stock constituents znso . sub . 4 . 7h . sub . 2 o 3 g / l feso . sub . 4 . 7h . sub . 2 o 10 g / l mnso . sub . 4 . 4h . sub . 2 o 3 . 2 g / l cuso . sub . 4 . 5h . sub . 2 o 0 . 079 g / l h . sub . 3 bo . sub . 3 1 . 5 g / l ki 0 . 2 g / l na . sub . 2 moo . sub . 4 . 2h . sub . 2 o 0 . 5 g / l cocl . sub . 2 . 6h . sub . 2 o 0 . 56 g / l______________________________________ the trace elements were added to demineralised water , acidified with 35 ml / l of 98 % h . sub . 2 so . sub . 4 . * 20 g sucrose / l was added to the batch medium at the 20 l seed fermenter stage . any convenient method of sterilisation may be used , as may any depyrogenation method , for example ultrafiltration . the vitamins were always filter sterilised . table 2______________________________________mw11d medium______________________________________constituents batch medium feed medium______________________________________ saltskh . sub . 2 po . sub . 4 4 . 66 g / l 9 . 54 g / l mgso . sub . 4 . 7h . sub . 2 o 0 . 98 g / l 2 . 02 g / l cacl . sub . 2 . 2h . sub . 2 o 0 . 10 g / l 0 . 21 g / l h . sub . 3 po . sub . 4 ( 85 % w / w ) 1 . 63 g / l 3 . 33 g / l vitamins ca pantothenate 68 mg / l 140 mg / l nicotinic acid 114 mg / l 233 mg / l m - inositol 68 mg / l 140 mg / l d - biotin 0 . 34 mg / l 0 . 70 mg / l thiamine . hcl 17 . 1 mg / l 35 mg / l trace element stock 10 . 2 ml / l 21 ml / l sucrose 0 * 500 g / l______________________________________ trace element stock constituents znso . sub . 4 . 7h . sub . 2 o 3 g / l feso . sub . 4 . 7h . sub . 2 o 10 g / l mnso . sub . 4 . 4h . sub . 2 o 3 . 2 g / l cuso . sub . 4 . 5h . sub . 2 o 0 . 079 g / l na . sub . 2 moo . sub . 4 . 5h . sub . 2 o 0 . 5 g / l cocl . sub . 2 . 6h . sub . 2 o 0 . 56 g / l______________________________________ the trace elements were added to demineralised water , acidified with 35 ml / l of 98 % h . sub . 2 so . sub . 4 . * 20 g sucrose / l was added to the batch medium at the 20 l seed fermenter stage . any convenient method of sterilisation may be used , as may any depyrogenation method , for example ultrafiltration . the vitamins were always filter sterilised . after the medium was added to the vessel , the operating temperature of 30 ° c . was set , as well as the minimum stirrer speed , typically 400 - 500 rpm . the initial ph was adjusted with ammonia solution ( specific gravity 0 . 901 ) using a ph controller set at 5 . 7 ± 0 . 2 . 2m h . sub . 2 so . sub . 4 was also used as a ph corrective agent . sucrose to 20 g / l , mw10 batch vitamins , and breox fmt30 antifoam to 0 . 04 g / l are added to the vessel . sterile filtered air was introduced into the vessel at 0 . 5 vvm ( ie 0 . 5 liter non - compressed air per liter of medium per minute ), the medium was inoculated to 12 ± 2 mg cell dry weight l - 1 from an axenic shake flask culture and the mfcs computer system was initiated . following completion of the batch phase of growth ( signalled by a dissolved oxygen tension increase of & gt ; 15 % in 30 min ), addition of the feed medium was initiated , under control of the mfcs system . the control strategy was effectively the same as described below for the production fermenter . during the fermentation the airflow was increased in two steps in order to maintain a flow of approximately 1 vvm . further breox fmt30 was added to a final concentration of 0 . 3 g / l . the dissolved oxygen tension ( dot ) was controlled at 20 % air saturation by changing the stirrer speed . once the stirrer speed could be increased further and the airflow rate reached its maximum value , the feed control algorithm ( see below ) controlled the feed rate such that the dot did not decrease below 15 % in order to prevent oxygen limited conditions that , otherwise , would lead to formation of fermentation products . also rq was used as a feedback for the feed addition control . the feed rate was reduced every 10 min while rq ≧ 1 . 2 . moreover , a 120 min rq average ( rqavg 120 ) was calculated to filter the noisy rq signal ( goodey el al , 1996 ). the feed rate was reduced once every two hours by 20 % if the value of rqavg 120 ≧ 1 . 13 . due to an expected high rq value at the start of a fermentation this rqavg 120 control was not performed during the first 4 hours of the feed addition phase . at the end of the feed , the culture was transferred to a production vessel . production fermentation . the production fermenter ( fig8 ) was inoculated with the culture grown in the seed fermenter ( see above ). the cell dry weight ( cdw ) concentration in the seed fermenter was normally greater than 80 g / l . the cdw concentration in the production fermenter just upon transfer of the seed fermenter culture was 0 . 25 - 1 . 00 g / l . although it is preferred to initiate feeding within one hour , it can be delayed if necessary . the feed regime was intended to minimise the accumulation of ethanol and acetate , so as to maximise the cell and product yield . the fermentation was carried out in a fermenter such as that shown in fig8 designed to give optimum gas dissolution and bulk mixing . the fermenter was equipped with ports for , amongst other things , supplying feed medium , withdrawing medium at the end of the fermentation and introducing a probe for measuring electrical conductance . the vessel , which was subjected to a hot naoh wash and pfw rinse , contained one volume of sterile mw10 ( table 1 ), batch salts and trace elements . this medium may be sterilized independently of the vessel either by heat or filter sterilisation . it has been found in accordance with the present invention that it is advantageous for the fermentation medium , such as mw10 , to be free of ethylene diamine tetraacetic acid ( edta ), or a salt thereof , since its presence results in a significantly higher degree of coloured contaminants in the albumin produced . the operating temperature was set at 30 ° c ., and the stirrer speed regulated to be sufficient to maintain a homogeneous solution , typically about 50 rpm . the initial ph was adjusted with ammonia solution ( sg 0 . 901 ) ( controller set to 5 . 7 ± 0 . 2 ). 2m h 2 so 4 nay be used as a second ph corrective agent . the mw10 batch vitamins were added , as was a suitable antifoam , as required ( eg breox fmt30 to 0 . 4 g / l ). when the feed is started , the rq over - ride control was disabled until our and cer values are sufficiently high to make control effective ; the feed rate was reduced manually during this period if rq was consistently & gt ; 1 . 2 . the ph of the culture was kept constant at 5 . 5 by automatic addition of 17 % ( w / v ) ammonia . the temperature was kept at 30 ° c . sterile airflow was introduced at 0 . 5 vvm . during the fermentation the airflow was increased in three steps in order to maintain a flow of approximately 1 vvm . this was measured by a continuous mass spectrometric analysis ( fisons vg gas analyser ). the fermentation was then run as above . also the pressure in the fermenter was increased during the fermentation to approximately 0 . 5 bar g by using a brooks pressure controller . the feed rate was started at a feed rate , fr start , that was necessary to achieve a growth rate of approximately 0 . 07 h - 1 . then the feed rate was increased , by computer control , according to the algorithm : counter : a counter variable started at 0 and was increased by 0 . 0167 once every min . however , the counter variable was decreased : a . by 0 . 0167 once every min if the dissolved oxygen tension ( dot ) was less than 15 %. c . by 0 . 223 / k ( resulting in a 20 % feed rate reduction ) once every two hours while rqavg 120 ≧ 1 . 13 if the feed addition was started more than 4 h ago . the result of such a fermentation is shown in fig1 . it can be concluded that the conductance trend in general sloped downwards during the course of the fed - batch fermentation . the electrical conductance during a phase where the feed rate was suddenly increased by 20 % in order to establish the use of the conductance signal in the prevention and correction of acetate accumulation , a deliberate sudden step - increase of feed rate of 20 % was applied at some stage in a carbon - limited fed - batch fermentation similar to the one described in example 1 . the results are shown in fig2 . it is shown that the conductance increased significantly during the period where the over - feed was applied . in fact , the rq , a parameter often used in the control of bakers &# 39 ; yeast production , did not show a significant increase . this shows the usefulness of the conductance signal because acetate production is undesirable during bakers &# 39 ; yeast production . the increase in conductance correlated with an increase in acetate concentration in the culture as assayed in culture samples . the acetate was assayed using an enzymatic assay kit no . 148 261 from boehringer mannheim . the electrical conductance during a phase where the feed rate was suddenly increased by 40 % in a similar experiment as shown in example 2 a sudden 40 % feed rate increase was applied ( see fig3 ). the effects were more extreme than in example 2 , as would be expected . also the rq increased . however , a value of 1 . 2 , which typically is used as a level to instigate feed rate reductions ( see example 2 ), was not reached . this again shows that conductance is a more sensitive physical control parameter than rq . the use of a feed rate control algorithm incorporating electrical conductance in fig4 a flow diagram is shown representing the feed addition control algorithm that was used in this example . the basis was the normal control algorithm as shown in example 1 . the condition where an airflow or pressure set point increase prevents the conductance feed control to be applied for 1 hour was necessary due to the fact that airflow and pressure increases will result in a small increase in conductance due to changes in gas holdup volume . moreover , in comparison with example 1 the following additions were made to the feed rate control algorithm . the change in conductance ( δc in ms ) was measured over a time interval of 30 min . if the feed had been started within the last 1 . 5 h no feed back control would result . however , after that , in cases where the increase δc was ≧ 0 . 1 ms over the chosen time interval , an automatic feed rate reduction would result . the actual size of the feed rate reduction was made dependent on the actual value of δc as follows : fr reduced = f original *( 1 - δc ). no feed rate reduction would be applied if rq ≦ 0 . 95 or if the difference in rq 30 ( rq averaged over 30 min ) over a time interval of 20 min : rq 30 - rq 30 20 min ago ← 0 . 025 . both these conditions indicate that the yeasts were already co - metabolising the feed substrate and fermentation products , thus abolishing the need for feed rate reductions . an experiment was carried out where the exponential constant k ( see example 1 ) was set to 0 . 12 h - 1 which is so high that production fermentation products would be expected for this yeast strain which was the same as in example 1 . this was done to test the action of the control algorithm as shown in fig4 and explained above . the results are presented in fig5 . the figure shows a steady increase of conductance correlating with an increase in the acetate concentration . at 2 . 3 h ( batch age ) an automatic feed rate reduction was applied . this , however , was not sufficient and another automatic feed rate reduction was applied at 4 . 5 h ( batch age ). after that the acetate concentration reduced to 0 mm . then the acetate concentration increased temporarily at batch age 5 h , whilst the conductance was decreasing . at the same time an excess of ammonium ions , which will have been added in the period up to 4 . 5 h ( batch age ) for ph control , was probably being consumed as judged by the ph changes in the culture . it is known that ammonium ions conduct electricity better than acetate ions ( owens , 1985 ) which explains the overall decrease of the conductance signal . again a small peak in acetate concentration occurred at batch age 6 h . in this case the conductance increase was not enough to invoke a feed rate reduction . however , as judged by the reduction of the acetate concentration after that , further feed rate reductions were not necessary . the use of a feed rate control algorithm incorporating electrical conductance with the bacterial strain e . coli the bacterial strain escherichia coli dh5α was grown in a fermenter using the medium described by riesenberg et al ( 1991 ). the same control algorithm was used as in example 4 . however , the factor k was set at 0 . 4 h - 1 . in fig6 the result of the action of the control algorithm is illustrated . after a build up of acetate two automatic feed rate reductions resulted in a decrease of acetate from 45 to 5 mm . this artificially high challenge to the fermentation showed that the system would work even under extreme conditions . this represents a more realistic ( but still artificial ) challenge to the equilibrium of a fermentation . the bacterial strain e . coli dh5α was grown in a fermenter using the medium described by riesenberg et al ( 1991 ). a similar control was used as in example 4 . however , the factor k was set at 0 . 1 h - 1 . this would , under normal aerobic conditions , not lead to the production of organic anions . then between the batch age 21 . 3 - 22 . 3 h ( see fig7 ) the feed rate was increased manually in three steps . following this intervention , the conductivity increased and the feed rate was controlled according to the algorithm described in fig4 with the following modifications . a control step was taken once every 10 min ( as the conductivity increase was very steep ) but the size of the feed rate reduction was a quarter of that described in fig4 . thus the formula for feed rate was fr reduced = fr original ( 1 - δc / 4 ). as shown in fig7 this controlled the fermentation such that the acetate produced was consumed by the cells . this example shows that the control algorithms may be optimised for different situations such as different organisms , growth rate and media types . clarke p . m ., collins s . h . and mead d . j . 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