METHOD OF MONITORING FERMENTATION

Abstract: A method of monitoring fermentation in a fermentation broth. The method is conducted in such a way that the total number of particles in a sample from the fermentation broth is determined by means of a device giving an optical density reading which is a measure of the total number of particles present, that said sample is mixed with an excess of labelled specific antibodies directed against a product chosen from the group consisting of an inoculum, a desired fermentation product and an important contaminant, that the number of particles consisting of product bound to the labelled specific antibody is determined with the aid of the label used and is compared to the determined total number of particles, and that this comparison indicates if any particles contaminating the fermentation are present in the fermentation broth. The method can be performed in a continuous manner, and with computer assistance and the use of a FACS, also automatically.

Description: METHOD OF MONITORING FERMENTATION The present invention is concerned with the moni¬ toring of fermentations in any effected fermentation procedure. Background The difficulties in monitoring fermentations are widely recognised. In attempts to determine if the fermentation is proceeding adequately, if the inoculum is faultless and if the fermentation is conta¬ minated by contaminants, the efforts so far have been concerned with the identification of contaminants. This has usually comprised culturing samples from the fermentation broth and identifying any contami¬ nants. Since culturing naturally takes time the fermen¬ tation, which does not wait for results but continues, is lost from time to time before suitable measures can be taken to save it. In some fields, this happens quite frequently, and enormous amounts of money are wasted. Thus there is a need for a method of monitoring fermentations in a relatively short period of time so that suitable measures can be taken to save the fermentation. Short description of the invention In contrast to the prior art methods of monitoring fermentations the method of the invention is directed to the monitoring of a product chosen from the group consisting of an inoculum, a desired fermentation product and an important contaminant, and this is accomplished with the aid of labelled specific anti¬ bodies directed against the product which is monito¬ red. The method of monitoring fermentation in a fer¬ mentation broth according to the invention is cha¬ racterised in that the total number of particles in a. sample from a fermentation broth is determined by means of a device giving an optical density reading, which reading is a measure of the total number of particles present, that said sample is mixed with an excess of labelled specific antibodies directed against a product chosen from the group consisting of an inoculum, a desired fermentation product and an important contaminant, that the number of particles consisting of product bound to the labelled specific antibody is determined with the aid of the label used and is compared to the determined total number of particles, and that this comparison indicates if any particles contaminating the fermentation are present in the fermentation broth. The products which can be monitored by the method of the invention, can be divided into three main cate¬ gories, viz. cells ^ , e.g. the inoculum used, extra¬ cellular fermentation products and intracellular fer¬ mentation products (e.g. when recombinant DNA technique is used and the desired fermentation product is ex¬ pressed within cells). When the product which is monitored is a cell, the specific antibody used is directed against surface antigens of the cell. When the product which is monitored is an intra¬ cellular fermentation product, a detergent or chemical is used for cell disruption to free the product before labelled specific antibody directed against the product is added. The important contaminant can be important be¬ cause, for example, it is expected, commonly occurring and/or its presence is acceptable only up to a certain concentration. The labelling of the specific antibody used is performed according to known procedures and the label used can be an enzyme marker, radioactive isotope or a fluorescent marker of types already used in the field of immunoassay. Brief description of the drawing The drawing is a schematic representation of elements in a continuous monitoring line used in pre- ferred embodiments of the invention. Detailed description of the drawing The drawing comprises the following elements: A fermentation tank 1, a first device for sepa¬ ration of particles 2, and second pump 3, a first mixing coil 4 or 5 , a first device giving an optical density reading 6, a second reservoir 7, a third pump 8, a second mixing coil 9, a fourth reservoir 10, a fifth pump 11, a fourth mixing coil 12, a fourth pump 13, a valve 14, a FACS or a flow cytofluorimeter 15, a first reservoir 16, a first pump 17, a second device for separation of particles 18, a second device giving an optical density reading 19, a third reservoir 20, a fourth pump 21 and a third mixing coil 22. Detailed description of the invention The method of monitoring fermentation in a fer¬ mentation broth can, according to one embodiment of the invention, be conducted at different times during the fermentation, in the manner indicated below. A sample from the fermentation broth, taken from one or several sites in a fermentation tank, is optio¬ nally passed through a device for separation of par- . tides, such as a sonal separator or a filter having an appropriate pore size in accordance with the size of the particles consisting of the product which is to be monitored. The total number of particles in the sample is determined by means of a device giving an optical density reading, such as an UV spectro- photometer, which reading is a measure of the total number of particles present in the sample. Next, label- led specific antibody directed against the product which is monitored is added in excess to the sample, and the number of particles consisting of product bound to the labelled specific antibody is determined with the aid of the label used. How this is effected, is desribed below in connection with a preferred embo¬ diment of the Invention. The determined number of particles comprising the product which is monitored is now compared to the determined total number of particles in the sample. This comparison indicates If any particles contami¬ nating the fermentation are present in the sample and thus in the fermentation broth. The fermentation can thus be monitored by studying results of the above comparison taken at different times during the fermentation. The products which can be monitored by the method of the invention, can be divided into three main cate- gories, viz. cells, e.g. the inoculum used, extracel¬ lular fermentation products and intracellular fermenta¬ tion products (e.g. when recombinant DNA technique is used and the desired fermentation product is expres¬ sed within cells). When the product which is monitored is a cell, the specific antibody used is directed against chosen surface antigens of the cell. When the product which is monitored is an intra¬ cellular fermentation product, a detergent or chemical is used for cell disruption to free the product before labelled specific antibody directed against the product is added. The labelling of the specific antibody used is performed according to known procedures and the label used can be an enzyme marker, radioactive isotope or a fluorescent marker of a type already used in the field of immunoassay. A preferred embodiment of the invention comprises a method of monitoring fermentation in a fermentation broth conducted continuously in the following manner. The fermentation in the fermentation broth is conducted in any conventional fermentation tank (1) , under appropriate conditions. Continuous sampling from one or several sites in the fermentation tank (1) is effected, and the sample stream is optionally passed through a first device for separation of particles, such as a sonal separator or a filter (2) having an appropriate pore size (which can be varied depending on the size of the particles which are desired to pass) by means of a second pump ( 3 ) which pumps in the direction of the monitoring line. Optionally, the sample stream is passed through a first mixing coil arranged before ( 4) or after ( 5) the second pump (3) . The sample stream is next passed through a first device (6) giving an optical density reading (e.g. UV spectrophotometer ). The reading is used for determining the amount of diluent that is needed to give a constant number of particles in the sample stream. Diluent is added to the sample stream from a second reservoir (7) by means of a third pump ( 8 ) in an amount determined by the optical density reading. This amount can be determined by means of a computer, on the basis of the optical density reading, and the control of the third pump (8) can be effected by signals from the computer. The diluted sample stream is passed through a second mixing coil (9) and a liquid containing labelled specific antibody directed against a product, chosen from the group consisting of an inoculum, a desired fermentation product and an important contaminant, is added to the diluted sample stream in excess (based on the determined constant number of particles present in the stream) from a fourth reservoir (10) by means of a fifth pump (11). This addition of labelled specific antibody to the sample stream can be controlled by a computer with the aid of the reading for the total number of particles present, and the computer control- ling the fifth pump (11) pumping an adequate amount of said liquid containing labelled specific antibody to the sample stream. The sample stream is next passed through a second mixing and delay coil (12) where the product which is monitored, is interacting with the labelled specific antibody, and since the latter is present in an excess all the particles of the product which is monitored will be bound to labelled antibody. The sample stream is passed on to a fourth pump (13) which maintains a constant pressure in the monito¬ ring line, and if the pressure is exceeded an appropriate amount of the sample stream is discarded through a valve (14) which is activated at a pressure in the monitoring line exceeding a preset value. Again the maintaining of the constant pressure in the monitoring line can be accomplished by means of a computer con¬ trolling the fourth pump (13) and the valve (14). The sample stream discharged from the fourth pump (13) thus has a constant number of particles per unit of time, and with the aid of the label used the number of particles comprising the product which is monitored per unit of time can be determined and compared to the determined constant total number of particles present. This comparison indicates if any particles contaminating the fermentation are present in the sample stream and, thus, in the fermentation broth. Then measures to control the fermentation can be taken, e.g. to abort, freeze or treat the fermen- tation broth appropriately. The determination of the number of particles comprising the product which is monitored can be ef¬ fected in several known ways, depending of the label used. if the label used is a radioactive isotope, the sample stream containing both a free labelled specific antibody and a product-bound labelled specific antibody is analysed for free or bound labelled antibody by subjecting a sample from the sample stream to chroma- tographic separation or, preferably, by subjecting the sample stream continuously to chromatographic separation. Then either the amount of separated free labelled antibody or the bound counterpart is determined by means of a device giving a radioactivity reading (which is a measure of the number of particles com¬ prising the label). If the label used is an enzyme, a sample of the sample stream containing both a free labelled specific antibody and a product-bound labelled specific antibody is analysed for free or bound labelled antibody by e.g. enzyme linked immunosorbent assay (ELISA). If the label used is a fluorescent substance, which is preferred, then again a sample of the sample stream containing both a free labelled specific antibody and a product-bound labelled specific antibody is analysed for free or bound labelled antibody, e.g. by means of a fluorescence microscope or continuously by means of a flow cytofluorimeter or a fluorescence activated cell sorter (FACS). In the most preferred embodiment of the invention, a fluorescence activated cell sorter (FACS) is used °r continuous monitoring of the sample stream in the following manner. The sample stream from the fourth pump (13), which now has a known total number of particles present per unit of time, is fed, at a constant pressure, to a FACS (15). The FACS has a nozzle forming droplets, no at a constant rate, which droplets are passed through one or several laser beams, and the light emission from any particles passing the laser beam(s) is recorded from an angle of e.g. 90° to the laser beam(s), and the light scattering from the same particle is recorded from another angle of e.g. 150° from the laser beam(s), this light scattering giving an estimate of the size of the particle measured. The sample stream from the fermentation broth can now be monitored continuously from a display window of the FACS where e.g. the readings are sorted so that the relative size of the particles (abscissa) versus the relative degree of fluorescence (ordinate) are plotted in a system of coordinates. Thus the development of the fermentation can be monitored continuously by following the development of the product which is monitored in the display window. The product which is monitored is thus seen in the display window in one place according to its size and fluorescence, and any contaminants which are not fluorescent are seen close to the abscissa according to their size. Signals from the FACS can be transfered to a computer which can give alert signals or signals con¬ trolling the conditions of the fermentation. Optionally, reference particles bearing a flu¬ orescent marker of the same or different type as the one used for the specific antibody can be incorporated in the above monitoring line. The reference particles can be fed from a first reservoir (16) by means of a first pump (17) to the sample stream in the monitoring line somewhere before the fourth pump (13), preferably after the first device for separation of particles (2) and before the first mixing coil (4 or 5). The size of the reference particles is chosen to differ from the size of the particles consisting of the product which is monitored and which is bound to the labelled specific antibody. Thus the reference particles can be seen in a specific place in the display window of the FACS. If the reference particles move from their specific place, then something is wrong in the monitoring line, for example the pressure has dropped. When the product which is monitored is a cell, e.g. the inoculum, the sample stream from the fermen¬ tation tank (1) can be passed directly to the first device giving an optical density reading (6) by means of the second pump (3), optionally through the first mixing coil (4 or 5). A diluent from the second reser¬ voir (7) may or may not be added to the sample stream (8) by means of the third pump. If no diluent is added, then the amount of cells produced in the fermentation broth can be monitored by means of the FACS, and the optimum time for harvesting can be determined. When the product which is monitored is an extra¬ cellular fermentation product, then the sample stream from the fermentation tank ( 1 ) is first passed through the first device for separation of particles (2) to exclude, for example, cells from the sample stream. When the product, which is monitored is an intra- cellular fermentation product then the sample stream is passed by means of the second pump (3), optionally through the first device for separation of particles ( 2 ) , and/or the first mixing coil (4 or 5), to the first device giving an optical density reading (6), and from the second reservoir (7) a detergent or che¬ mical is added by means of the third pump (8) to the sample stream which is next passed through the second mixing coil (9) where cell disruption takes place. Then the stream is optionally passed through an appro¬ priate second device for separation of particles, such as a sonal separator or a filter (18) to clear the sample stream from cell debris and the total number of particles present in the sample stream is next determined by means of a second device giving an optical density reading (19) which is used for determining the amount of labelled specific antibody that is added to the stream from the fourth reservoir (10) by means of the fifth pump (11). If the particle size of the product bound to the labelled specific antibody is inconveniently small for the FACS sorting of particles, then the monitoring line outlined above can be supplemented with an addi¬ tional, third reservoir (20) containing particles bearing specific antibodies directed against the product which is monitored, a fourth pump (21) pumping said particles to the sample stream at a site before or after the site of the addition of labelled specific antibodies, in which case also an additional, third mixing coil (22) is inserted in the monitoring line between the sites for addition of particles bearing specific antibodies and labelled specific antibody, respectively. The whole monitoring line can be automated with the assistance of computers, and signals from the FACS can control the conditions for the fermentation. In appropriate utilisation of the method according to the invention, fermentation in a fermentation broth can be monitored continuously from the inoculum to the end. First, the start-up of the fermentation is moni¬ tored by following the development of the inoculum and checking that the fermentation broth is free from contaminants. Then the fermentation is monitored in respect of the desired fermentation product to check that the fermentation broth is free from contaminants. „ From time, to time no dilution of the sample stream is effected, and thus the amount of product produced can be followed with the aim to determine the optimum time for harvesting. The method of monitoring fermentation according to the invention can advantageously be utilised to monitor several products chosen from the group consisting of inocula, desired fermentation products and important contaminants simultaneously by means of a FACS, as long as they differ in particle size and/or fluorescence. Antibodies directed against some important, expected and/or commonly occurring contaminants will provide (together with the knowledge of their size) not only an alert signal that a contaminant is present, but also an early identification and determination of the concentration of this contaminant(s) , which then can be ' compared to existing recommendations etc. to abort, freeze or treat the fermentation broth appro¬ priately. When fermentation monitoring according to the present invention is effected by means of a FACS, an unknown contaminant which is discovered can be recovered in a purified state separately from the cultured organism, thus allowing for rapid testing of possible methods of controlling the situation. It will thus be obvious to the expert that the method according to the invention can be used for checking the purity of an inoculum before it is added to the fermentation broth, with the aid of labelled specific antibodies directed against the inoculum and preferably a FACS, an embodiment which is within the scope of the invention. The method of monitoring fermentation in a fermen- tation broth according to the invention can be used for controlling the conditions of fermentation op¬ tionally in a continuous manner.

Claims: CLAIMS 1. A method of monitoring fermentation in a fer¬ mentation broth, c h a r a c t e r i s e d in that the total number of particles in a sample from the fermentation broth is determined by means of a device giving an optical density reading which is a measure of the total number of particles present, that said sample is mixed with an excess of labelled specific antibodies directed against a product chosen from the group consisting of an inoculum, a desired fermentation product and an important contaminant, that the number of particles consisting of product bound to the labelled specific antibody is determined with the aid of the label used and is compared to the determined total number of particles, and that this comparison indicates if any particles contaminating the fermentation are present in the fermentation broth. 2. A method according to claim 1, c h a r a c ¬ t e r i s e d in that the total number of particles in the sample from the fermentation broth is determined by means of an UV spectrophotometer. 3. A method according to claim 1 or 2, c h a ¬ r a c t e r i s e d in that the label used is an enzyme marker, and that the determination of the number of particles consisting of product bound to the labelled specific antibody is effected by enzyme-linked immuno- sorbent assay (ELISA). 4. A method according to claim l or 2, c h a ¬ r a c t e r i s e d in that the label used is a radio¬ active isotope, and that the determination of the number of particles consisting of product bound to the labelled specific antibody is accomplished by subjecting the sample to chromatographic separation and the amount of separated free labelled antibody, or the bound counter¬ part is determined by means of a device giving a radio- activity reading. 5. A method according to claim l or 2, c h a r a c ¬ t e r i s e d in that the label used is a fluorescent substance, and that the determination of the number of particles consisting of product bound to the labelled specific antibody is effected by means of a fluorescence microscope, a flow cytofluorimeter or a fluorescence activated cell sorter (FACS). 6. A method according to claim 1 or 2, performed in a continuous manner, c h a r a c t e r i s e d in that the label used is a fluorescent substance, and that continuous sampling of the fermentation broth is effected from one or several sites in a fermentation tank ( 1 ) , that this sample stream is optionally passed through an appropriate first device for separation of particles (2), that optionally reference particles bearing a fluorescent marker are added to the sample stream from a first reservoir (16) by means of a first pump (17), that the sample stream is passed through a first mixing coil (4, 5) by means of a second pump (3), the first coil (4, 5) being arranged before (4) or after (5) the second pump ( 3 ) , that the sample stream is next passed through a first device (6) giving an optical density reading which is a measure of the total number of particles present in the sample stream and which is used for determining the amount of diluent, detergent or chemical to be added to the sample stream, that the determined amount of diluent, detergent or chemical is added to the sample stream from a second reservoir (7) by means of a third pump (8), that the sample stream is passed through a second mixing coil (9), that the sample stream is optionally passed through an appropriate second device for separation of particles (18), that the sample stream is optionally passed through a second device (19) giving an optical density reading which is a measure of the total number of particles present in the sample stream, that optionally particles bearing specific antibodies directed against a product chosen from the group consisting of an inoculum, a desired fermentation product and an important contaminant are added to the sample stream from a third reservoir (20) by means of a fourth pump (21), that optionally the sample stream is passed through a third mixing coil (22), that an excess of labelled specific antibodies directed against a product chosen from the group consisting of an inoculum, a desired fermentation product and an impor¬ tant contaminant are added to the sample stream from a fourth reservoir (10) by means of a fifth pump (11), that the sample stream is next passed through a fourth mixing and delay coil (12), that the sample stream is passed through a fourth pump (13) which maintains a constant pressure in the monitoring line with the aid of a valve (14) discarding a part of the sample stream at a pressure exceeding a preset value, that the sample stream discharged from the fourth pump (13) is passed to a fluorescence activated cell sorter (FACS) (15) which sorts the particles present in the sample stream according to relative particle size and relative degree of fluorescence, and that the sample stream from the fermentation broth thus is continuously monitored by means of the FACS (15). 7. A method according to claim 6, c h a r a c ¬ t e r i s e d in that the control of the monitoring line is computer-assisted. 8. A method according to claim 6 or 7, c h a r ¬ a c t e r i s e d in that several products chosen from the group consisting of inocula, desired fermentation products and important contaminants are monitored simul¬ taneously. 9: Use of the method of monitoring fermentation in a fermentation broth according to any one of the preceding claims, for controlling the conditions of fermentation. 10. Use of a method of monitoring fermentation in a fermentation broth, according to claim 6, 7 or 8, for continuous control of the conditions of fermen¬ tation.

Inventors: SVENSON STEFAN, SVENSON, STEFAN
Applicants: TRION FORSKNING & UTVECKLING, TRION FORSKNING- OCH UTVECKLINGS AKTIEBOLAG
Application year: 1987
Main classification: C12Q1
IPC codes: C12M1, C12Q1, G01N33