A PROCESS AND AN APPARATUS FOR THE RECOVERY OF A POLYPEPTIDE FROM A FERMENTATION BROTH

Abstract: In a process for recovering a polypeptide, such as an insulin-like material or hGH, from a fermentation broth, the fermentation broth is passed through a bed of a chromatographic material containing hydrophobic groups, such as phenyl sepharose, and then the adsorbed insulin material is eluted with an aqueous medium, which, if desired, may contain a water miscible organic solvent. When the chromatographic separation is carried out under such conditions, the polypeptide is isolated in a high yield in a single step from the fermentation broth and the other components contained in the broth. Further, a high concentration factor is obtained. An apparatus for carrying out the process consists of a fermentation container (1) and at least one chromatographic column (8, 9) connected with the container, as well as means (2, 7, 10) for circulating the fermentation broth from the fermentation container (1) through the chromatographic column or columns (8, 9).

Description: A process and an apparatus for the recovery of a poly¬ peptide from a fermentation broth. The present invention concerns a process for recovering a polypeptide from a fermentation broth. Polypeptides, which were previously recovered from ani¬ mal tissue or organs, e.g. by extraction, are now increas¬ ingly produced by bio-technjcal processes using micro¬ organisms, such as bacteria or yeast cells, transformed by engineering in a manner such as to form the desired polypeptide as a fermentation product. In this way it has been possible to produce biosynthe- tic insulin, hGH, interferoπ, so atotropin, prolactin and many other desired polypep ides. In fermentation of a substrate by means of the transformed microorganisms the desired polypeptide is in certain cases secreted to the medium. Here, the polypeptide will occur in a low concentration, e.g. 10-200 mg/1, and will be mixed with proteolytic enzymes which have simultaneous¬ ly been secreted from the microorganism. The fermenta- tion is usually carried out at a relatively high tempera¬ ture, such as from 25-40°C, with a consequent risk of pro¬ teolytic degradation as well as deπaturation of the poly¬ peptide. It would therefore be desirable to isolate the polypeptide from the fermentation broth in statu nascen- di at a low temperature before the purification proper took place, but owing to the composition of the fermen¬ tation broth (high conduc ivity, presence of microorga¬ nisms) purification proper cannot be started in the known processes until the microorganisms have been removed and a buffer change has taken place. Owing to the relative¬ ly small concentrations of the desired peptide it is ne- cessary, on an industrial scale, to treat extremely large liquid volumes, so that many methods used on a smaller scale are inapplicable or too expensive. It is known to separate the fermentation broth with the desired polypeptide from the microorganisms by centri¬ fugation, filtration or aqueous two-phase extraction. The normal procedure is then to reduce the volume to obtain a concentration of the polypeptide, enabling chromatogra¬ phic purification, such as 1 - 1 % w/v. When an optional buf¬ fer change is made as an intermediate step, purification proper can be started by either gelfitration, ion exchan¬ ge chromatography, affinity chromatography, hydrophobic chromatography or chromatofocusiπg . All these methods are extensively used for purification of biological substances. On a large scale, however, the economy plays an important role, so the number of unit operations should be minimized. Thus, isolation of the desired polypeptide from the fer¬ mentation broth is desirable - preferably by a continuous and gentle process comprising a minimum of unit operations, and so that the isolated product is present in a form which lends itself to further purification. The present invention is based on the finding that the po¬ lypeptide can be extracted directly from the fermentation broth under specific conditions, if desired even during the very fermentation. Thus, the invention concerns a process for recovering a polypeptide from a fermentation broth, said polypeptide having been formed by fermentation with a microorganism, said fermentation broth being treated with a chromato¬ graphic material containing hydrophobic groups and adsorb- ing the polypeptide contained in the fermentation broth, and the process is characterized by passing the fermenta¬ tion broth with its content of polypeptide and optional¬ ly also the microorganism directly through a bed of the said chromatographic material containing hydrophobic groups, and then eluting the polypeptide adsorbed on the chromatographic material with an aqueous medium which, if desired, may contain water miscible organic solvent. When the process is carried out as stated, it is possible, in a single step, to recover the desired polypeptide di¬ rectly, with a high yield and a high concentration fac¬ tor. Owing to its process-technical simplicity the pro¬ cess is very advantageous. Thus, the desired polypeptide is obtained in the eluate in a high yield and great puri- ty, so that the subsequent purification can be performed without complications. According to a preferred embodiment of the invention, the fermentation broth is treated continuously with the micro¬ organism in a suspended state and during the very fermen¬ tation, the broth being circulated through the bed of the chromatographic material, expediently in a fluidized state, and recycled for further fermentation. The continuous re¬ moval of the fermentation product, i.e. the desired poly¬ peptide, reduces the risk of degradation, thus giving an optimum yield. This effect can be intensified if the broth is kept cooled to 1-12°C, preferably 4-6°C, during the cir¬ culation. The process of the invention has been found to be useful in the recovery to proinsulin or other insulin-like material, called ILM, from a fermentation broth formed by cultivation of a correspondingly transformed yeast cell. Even small amounts of ILM in a very low concentration can be isolated in a yield high and with a high concentration factor, without any special requirements of the fermenta- tion medium. Thus, in the present process it has been found possible to obtain a concentration factor in excess of 100, e.g. typically 100-1000. This gives a solution of ILM which is considerably freed of the other components of the fermentation broth. In addition, application at pH<4 is possible, so that the activity of the proteoly- tic enzymes is impeded. Appropriately, before the treatment with the chromatogra¬ phic material, the fermentation broth is admixed with ions having a high protein salting-out effect in a concentra¬ tion of 0.01 - 1 molar, preferably 0.05-0.4 molar. Examples of suitable ions are P0. 3-, SO. 2-, CCrH-U,COO NH Na and K . Preferred salts releasing these ions are.NH. ) 2 S0 4 and (NH 4 ) 3 P0 4 and K-.P0 4 , CH 3 C00NH 4 . Human growth hormone, hGH, has been recovered in a cor¬ responding manner from a fermentation broth formed by cul¬ tivation of a substrate with a microorganism. The chromatographic material must have a suitable speci¬ fic affinity to the desired polypeptide. Examples of chro- matographic materials suitable in connection with ILM or hGH are sepharose containing aryl or alkyl groups, e.g. octyl sepharose and phenyl sepharose. ILM binds so strong¬ ly to these gels that it is not necessary to add ions hav¬ ing protein salting-out effect to the fermentation broth. Phenyl sepharose is the preferred chromatographic material for the recovery of ILM since this protein binds suit¬ ably strongly, without rendering the subsequent elution di fficult . An eluate is formed in the process of the invention, con* taining polypeptides in a concentrated form, and the de¬ sired polypeptide can be isolated from the eluate in a pure state in a manner known per se. The invention also concerns an apparatus for carrying out the process of the invention, said apparatus being characterized by the features defined in the character-, izing portion of claim 10. The invention will be explained more fully below with re¬ ference to the drawing, which shows a process diagram or a system for isolation of polypeptide from a fermentation broth. The apparatus comprises a fermentation container 1 which can be supplied with substrate or nutrient fluid from a supply tank 15 through a supply pipe 16. The amount of supplied nutrient fluid can be adjusted by means of a valve 16a. A pipe 2 leads from the container 1 through a pump 3 to an ultrafiltration apparatus 5. A conduit 6 for retentate <_aleads back to the tank 1,' and a conduit 7 for filtrate leads to two columns 8, 9 containing chromato¬ graphic material. A (not shown) heat exchanger may be in¬ serted in the conduits 2 and 6 to recover heat, if the temperature in the ultrafiltration apparatus 5 has been adjusted considerably lower than the temperature of the fermentation broth in the container 1. To remove excess of yeast cells formed in the fermenta¬ tion container 1, conduits 17, 19, 22 connect the contai¬ ner with a pump 18 and a centrifuge 20, which is provided with a drain pipe 21 for yeast concentrate. The columns 8, 9 are connected with a tank 11 for equili¬ bration liquid or elution liquid, which can be passed to one or the other of the columns 8, 9 by adjustment of a change-over valve 12 and valves 12a and 12b. By opening of a valve 13a or 13b, the eluate can be passed through a pipe 13 to a storage tank 14 for eluate. The invention is illustrated by means of the following examples, where examples 1 and 2 concern the operation of the embodiment of the apparatus of the invention which is shown in the drawing. EXAMPLE 1 10 1 of human proinsulin-producing yeast culture (Saccha- romyces cerevisiae AB 103-1 containing the plasmid py-BCA 5) were cultivated to an optical density of 3.5, measured at 610 nm in the fermentation container 1. The fermentation broth was passed through an ultrafiltra¬ tion system 5 of the Millipore Pellican Casette Svstem type with an 0.5 μ diaphragm. The retentate, consisting of a concentrated suspension of the yeast cells, was re¬ cycled through the pipe 6 to the fermentation container 1. The filtrate, a clear yellowish liquid containing insu¬ lin-like material (c = 5 μq/tcil was transferred at 4°C through the pipe 7 to a column 8 from Pharmacia, diameter 2.5 cm, height 10 cm, packed with phenyl sepharose CL6B, equilibrated with a buffer, 0.1 M (NH, ) ? S0. adjusted to a pH of 8.0. The eluate from the column 8 was drained off through the pipe 13 as waste. No insulin-like material could be detected in the eluate. After the volume of the fermentation broth had been reduced to 0.5 1, the pump 3 was stopped, and the column 8 was flushed with 2 column volumes of buffer from the tank 11. 50?ό v/v ethanol u/as introduced into the tank 11, and then the column 8 was eluted with this buffer, a total of 2 column volumes. The eluate was collected in the storage tank 14. Yield 87?ό w/w with a concentration factor of 100. EXAMPLE 2 25 1 of a human proinsulin-producing yeast culture (Sacc- haromyces cerevisiae, AB 103-1 containing the plasmid py- BCA 5) were cultivated to an optical density of 4.3 at 37°C, measured at 610 n in the fermentation container 1. The fermentation broth contained human semi-synthetic insulin in a concentration of 100 mg/1. During continuous operation and after cooling to 4°C, the fermentation broth, including yeast cells and insulin, was passed through an ultrafiltration apparatus 5 of the Pellicon Casette System type with an 0.5 μ diaphragm and recycled to the fermentation container 1. The filtrate containing semi-synthetic human insulin in a concentration of 100 mg/1 was passed via the conduit 7 to a column 8 (2.5 x 10 cm from Pharmacia, packed with phenyl sepharose CL6B, the column having been equilibrated beforehand with a buffer, 0.05 M (NH. ),PC , pH = 8.0 at 4°C. The eluate from the column 8 was recycled via the conduit 10 to the fermentation container 1. After 5 hours' cyclic operation the process was repeated, now with the column 9 inserted. This column was designed and equilibrated in all respects like the column 8. The column 8 was now flushed with a volume 0.05 M (NH. ),P0 , pH 8.0, and then the bound protein was eluted with 50?ό v/v ethanol at 4°C, a total of 150 ml. The eluate was collected in the storage tank 14. The column 8 was reequi' librated as stated above. The above-mentioned change between the two columns 8 and 9 was repeated for a total of 4 times. The overall volume in the storage tank 14 was measured to 1.2 1 with an insulin concentration of 1.8 mg/ml, corresponding to a recovery percentage of 88. EXAMPLE 3 525 ml of Saccharomyces cerevisiae pAB18, in which 5 mg of biosynthetic human growth hormone had been dissolved, were adjusted with phosphoric acid and 25?ά ammonia water to 0.3 M and pH = 6.85. The yeast suspension - cultivated to an optical density of 16.6 - was applied from below and up through a 1.6 x 5 cm column, containing suspended phenyl sepharose pa rt icles to form a fluidizing bed. Amount of flow 400 ml/hour. Temperature 20°C The column had been equilibrated before¬ hand with 0.3 M ammonium phosphate, pH 8.0. The applica¬ tion was performed as a cylic process during 16 hours, and then flushing was effecting with 65 ml of an equ--.li- bration buffer. The top piston was then pressed down to the surface of the gel, and elution was performed with 100 ml of H„0 in a direction from top toward bottom. The amount of hGH present in the application liquid, flush¬ ing liquid and eluate were determined by RP-HPLC. The application liquid contained 5 mg of BhGH, the flus¬ hing liquid contained 0.2 mg of BhGH, and the eluate con¬ tained 4.8 mg of hGH. 96?ό of the present hGH was thu-s: eluted in an aqueous solution without yeast cells with a con¬ centration factor of 5. EXAMPLE 4 4 1 of an ILM-producing yeast culture (Saccharomyces ce¬ revisiae AB 103-1 containing the plasmid py-BCA5) were cultivated to an optical density of 3.5, measured at 610 nm . Ultrafiltration was performed on a Millipore ultrafil¬ tration equipment Pellicon Casette System with an 0.5 u diaphragm. The clear yellowish fermentation broth con¬ taining insulin-like material (c = 0.5 ^g/ml) was adjusted at 4°C to 0.1 M (NH )„S0 4 and adjusted to a pH of 7.2. The broth was applied at 4°C to a column from Pharmacia, diameter 5.0 cm, height 15 cm, packed with phenyl sepha¬ rose CL 4E , equilibrated with a buffer 0.1 M (NH ή ) S0 4 , adjusted to a pH of 7.2. Elution of the insulin-like ma- terial during application could not be detected. After completed application, flushing was performed with 3 co¬ lumn volumes of an equilibration buffer, followed by elu¬ tion of the insulin-like material with H„0. Yield 85 % by weight with a concentration factor of 100. EXAMPLE 5 200 ul of pork insulin, dissolved in 0.05 M ammonium phos¬ phate, pH = 8.2 (c = 0.94 mg/ml ) , were applied to an 0.6 x 5 cm column, packed with phenyl sepharose CL-6B FF , equilibrated with 0.05 M ammonium phosphate, pH = 8.2. After completed application, flushing was performed with 5 column volumes of an equilibration buffer, and then the insulin was eluted with 7M urea. Yield 92 % by weight. EXAMPLE 6 10.2 ml of fermentation broth py-BCA5 containing pork in- sulin, c = 20 ^μg/ml, were admixed with 150 μ l of H--P0. (ortho-) to pH = 2.0. The solution was applied to an 0.6 x 5 cm column, packed with phenyl sepharose ^ CL4B, equilibrated with 0.05 M ammonium phosphate, pH = 8.2. After completed application, flushing was performed with 5 column volumes of an equilibration buffer, and then the insulin was eluted with H„0. EXAMPLE 7 10.2 ml of fermentation broth pyBCA5 containing pork in¬ sulin, c = 10 jjg/ l, were admixed with 150 ^ul of H,P0 4 to pH = 2.1. The solution was applied to an 0.6 x 5 cm column, packed with phenyl sepharose CL6B FP^, equilibrated with 0.1 M ammonium acetate, pH = 8.2. After completed application, flushing was performed with 5 column volumes of an equilibration buffer, and then the insulin was eluted with H„0. EXAMPLE 8 10.1 ml of fermentation broth pyBCA5 containing pork in¬ sulin, c = 10 were admixed with 150 μ l of acetic acid. pH was adjusted to 8.0 with 12.5?ό NH,. The solution was applied to an 0.6 x 5 cm column packed with phenyl sepharose CL4B (__), equilibrated with 0.1 M po- tassium a-phosphate , pH = 8.2. After completed applica¬ tion, flushing was performed with 5 column volumes of an equilibration buffer, and then the insulin was eluted with H ∑ 0. EXAMPLE 9 1 mg of pork insulin dissolved in 10 ml of 0.3 M H,P0, 3 4 (-ortho), pH = 3.0 with 12.58 NH,, was applied to an 0.6 x 5 cm column packed with phenyl sepharose CL4B R, equili- brated with 0.3 M ( H 4 ) 3 P0 4 , pH = 8.0. After completed application, flushing was performed with 5 column volumes of an equilibration buffer, and then the insulin was eluted with H_0. Yield 93?ό by weight. EXAMPLE 10 500 ml of an ILM producing yeast culture (Saccharomyces cerevisiae pAHlδ containing the plasmid pYBCA5), in which 50.1 mg of pork insulin had been dissolved, were adjusted with phosphoric acid and 25?ό ammonia to a concentration of 0.3 M and a pH of 3.0. The yeast suspension - cultivated to an optical density of 17 - was applied from below and up through a 1.6 x 5 cm column containing suspended phenyl sepharose particles to form a fluidized bed. The aolumn had been equilibrated beforehand in 0.3 M ammonium phosphate, pH = 8.0. Af¬ ter application, flushing was performed with 65 ml of an equilibration buffer, and then the flow was stopped to sediment the gel. The top piston was then pressed down to the surface of the gel, and a column volume of H ? 0 was applied in the direction from top toward bottom. The flow was then stopped for 30 minutes, followed by elution with 25 ml of H ? 0. The eluate was a clear, slightly yellowish insulin solu¬ tion without yeast cells. The amount of insulin present in the application liquid and eluates was determined on RP-HPLC. In the applica¬ tion, 25.5 mg were bound to the column, and then the co¬ lumn was apparently saturated. During the flushing pro¬ cess an amount corresponding to 2.9 mg was detected in the eluate. Then 25.0 mg of insulin were elu ted with H-0.

Claims: P a t e n t C l a i m s : 1. A process for recovering a polypeptide from a fer¬ mentation broth, said polypeptide having been formed by fermentation with a microorganism, said fermentation broth being treated with a chromatographic material containing hydrophobic groups and adsorbing the polypeptide contained in the fermentation broth, c h a r a c t e r i z e d by treating the fermentation broth, optionally after re¬ moval of the microorganism, with the said chromatographic material containing hydrophobic groups, and eluting the polypeptide adsorbed on the chromatographic material with an aqueous medium, which if desired, may contain a water miscible organic solvent. 2. A process according to claim 1, c h a r a c t e r - i z e d by continuously circulating the fermentation broth with the microorganism in a suspended state from a fer¬ mentation section through a fluidized bed of the chroma¬ tographic material, and then recycling the microorganism suspension to the fermentation section. 3. A process according to claim 1, c h a r a c t e r ¬ i z e d by circulating the fermentation broth through an ultrafiltration apparatus to separate the retentate con¬ taining the microorganism, recirculating the retentate containing the microorganism, and passing the filtrate through a bed of the chromatographic material, and, if desired, recycling it as a substrate. 4. A process according to any of claims 1-3, c h a r a c t e r i z e d in that the microorganism is a yeast cell transformed by introduction of a gene which codes for the desired polypeptide. 5. A process according to claim 4, c h a r a c t e r ¬ i z e d in that the desired polypeptide is human proinsulin. 6. A process according to claim 4, c h a r a c t e r ¬ i z e d in that the desired polypeptide is hGH. 7. A process according to any of claims 1-6, c h a r a c ¬ t e r i z e d in that the chromatographic material is phe¬ nyl sepharose, and that the eluting agent is a mixture of water and ethanol. 8. A process according to any of claims 1-7, c h a r a c - t e r i z e d by adding ions with a high protein salting- out effect in a concentration of 0.01-1 molar, preferably 0.05-0.4 molar, to the fermentation broth before the treat¬ ment with the chromatographic material. 9. A process according to claim 8, c h a r a c t e r - i z e d by adjusting the pH of the fermentation broth to 3-5. 10. A process according to any of claims 1-9, c h a r a c e r i z e d by-keeping the fermentation broth cooled to a temperature of 2-12°C, preferably 4-6°C, during the treatment with the chromatographic material. 11. An apparatus for carrying out the process according to claims 1-10, c h a r a c t e r i z e d by consisting of a fermentation container (1) and at least one chroma¬ tographic column (8, 9) connected with said container, as well as means (2, 7, 10) to circulate the fermentation broth from the fermentation container (1) through the chro¬ matographic column or columns (8, 9). 12. An apparatus according to claim 11, c h a r a c t e r i z e d by comprising an ultra iltration device (5) connected with means (6) for recycling the microorganism suspension to the fermentation container (1), and means (7) for transferring filtrate to the chromatographic co¬ lumn or columns (8, 9).

Inventors: HEJNAES KIM RY, PEDERSEN JOHN, HEJNAES, KIM, RY, PEDERSEN, JOHN
Applicants: NORDISK GENTOFTE, NORDISK GENTOFTE A/S
Application year: 1985
Main classification: C12P21
IPC codes: C07K1, C07K14, C12M1, C12P1, C12P21, C12R1