WO2001057218A1

WO2001057218A1 - A process for producing purified biologically active, free form of recombinant human interferon gamma

Info

Publication number: WO2001057218A1
Application number: PCT/IB2000/000318
Authority: WO
Inventors: Sharad Kumar Vyas
Original assignee: Sharad Kumar Vyas
Priority date: 2000-02-01
Filing date: 2000-03-20
Publication date: 2001-08-09
Also published as: AU3185400A; IN187900B; EP1171603A1; BR0007186A

Abstract

The invention discloses a novel process for production of biologically active, 'free form' of recombinant human interferon gamma by cytoplasmic expression using Escherichia coli (E.coli) as the bacterial host and purification of the interferon gamma so produced.

Description

A PROCESS FOR PRODUCING PURIFIED

BIOLOGICALLY ACTIVE, FREE FORM OF

RECOMBINANT HUMAN INTERFERON GAMMA

FIELD OF THE INVENTION

The invention relates to a process for producing purified, biologically active, 'free form' of recombinant human interferon gamma (HIG) by cytoplasmic expression in Escherichia Coli, as bacterial host.

BACKGROUND OF THE INVENTION

The background of the invention has been illustrated with reference to the following prior patent publications either generally or specifically :

U. S. PATENTS

5,595,888 I 1997 I Gray et al

I 5,690,925 1997 ] Gray et al

OTHER FOREIGN PATENTS

Interferons are a group of proteins produced by the cells of most of the vertebrates and characterized by their ability to inhibit the growth of viruses exposed to these proteins. Interferon proteins have been classified into three types - alpha, beta and gamma based on physiological functions, antigenicity and structural differences. Interferon gamma has number of characteristics that differentiate it from alpha and beta forms of interferons. Among these differences are antigenic distinctiveness and greater activity with regard to immunoregulation and antitumor effects. Human interferon gamma may be produced by T-lymphocytes, stimulated by mutagens or by antigens to which they are sensitized. It may also be obtained through cloning and expression techniques well known to the art.

The lymphocyte route for the production of human interferon gamma involves costly chemicals (viz. fetal calf serum, micronutrients), fermenter design, and a quality check on the lymphocyte cell lines used (without any mycoplasma/viruses which need to be checked for every batch and stock). The quantity (yield) of HIG produced is very low, fermentation cycle is more than 15 days to harvest the broth for purifying the HIG. The infrastructure facility required for the production of HIG through the lymphocyte route is also cumbersome. Recombinant DNA technology has permitted the expression of exogenous or foreign proteins in bacteria and other host cells.

Builder et. al., (US Patent Nos.4,511,502 & 4,620,948) discloses a method of production of recombinant human interferon gamma by cytoplasmic expression using Escherichia coli as bacterial host. In this method, transformed E. coli K-12 cells were grown in broth containing 10.0-g/lit yeast extract, and 5.0 g/lit. tryptone to a cell density of 2-4 X 10⁸ cells/ml. 3-5% volume of this culture was inoculated into M9 medium (J H Miller, Expts. Molecular Genetics, page 431, Cold Spring Harbor Laboratory, 1972). The cultures were grown in fermenter with sufficient agitation and aeration to achieve a growth rate of 60 to 90 min per cell division; glucose was fed to the cultures to maintain growth, but did not exceed 50 g per liter during the fermentation, and the pH of the cultures was controlled at 6.8-7.2 by NaOH or (NH OH. At cell densities of 5-10 g dry weight per liter, indole acrylic acid (IAA) and indole propionic acid (IPA) was added to the cultures to a concentration of 25 to 50 mg per liter. 2 to 5 hrs after the addition of IAA or IPA, the E. coli cells became elongated and one or more retractile bodies (Interferon gamma in insoluble form) could be seen under phase contrast microscope.

Gray et.al. (US Patent Nos.5,574,137, 5,595,888, 5,690,925) also discloses production of recombinant human interferon gamma in a fermenter with a working volume of about 10 to 1000 lit. The fermentation is carried out at 25°C to 40°C under conditions of agitation (100 to 1000 rpm) and aeration (0.5 to 1.5 wm) at a pH between 6,5 to 7.5.

There are, however, several serious drawbacks to the cytoplasmic expression of recombinant proteins in Escherichia coli in the hitherto described prior art processes. The intracellularly synthesized proteins are only partially available in the 'free form', and remain mainly in the reduced state generally forming insoluble inclusion bodies. These proteins do not have the correct conformation and disulfide bridges are not formed. In order to obtain a native protein, the molecule has to be oxidized, and refolded to achieve the correct conformation for biologically active protein. Recovery for the refolded protein is low and these protein often displays heterogeneity and to some extent immunogeniciry. Thus, an expression and purification scheme for biologically active interferon gamma, which allows correct folding in vivo, is highly desirable. The subject invention surprisingly finds that expression by a combination of reduced temperature and low inducer concentration results in more than 15% 'free-form' of biologically active interferon gamma. Further, interferon gamma containing cells so obtained are liberated from the host cells by various means such as osmotic shock, ultrasonic vibration, grinding or by high shear disruption and then processed to isolate the interferon gamma. The insoluble debris is separated by centrifugation and the interferon gamma containing supernatant is collected for purification. Several procedures are described in the art of separating and purifying interferon gamma from the supernatant collected from the centrifugation step. The processes of purifying interferon gamma from cell free supernatant are disclosed in the references cited above in which an affinity column such as Concanavalin - A Sepharose is used, followed by chromatography on earboxymethyl silica column using an increasing salt gradient and finally on a silica gel permeation column. If sufficient purity is not obtained then concentration and chromatography on either the TSK (of TOSOH Corporation) or Carboxy Methyl column is used for further purification. These purification processes requiring a multitude of steps, which have caused degradation of interferon or aggregation of interferon molecule or otherwise resulted in interferon gamma product obtained in low yield or low activity. So, it would be also desirable to provide a purification scheme to separate interferon gamma from cell debris of the disrupted cells in which interferon gamma was produced free from cell contaminants in high purity and activity.

The expression "free-form" as used in description and claims of the instant application means the state of the protein namely human interferon gamma which exists in a non-aggregate, soluble form inside the cell in the cytosol. SUMMARY OF THE INVENTION

The invention provides a sequence of bioprocess steps for the production and purification of recombinant human interferon gamma in biologically active form.

The first object of the invention is to produce biologically active "free form" human interferon gamma in high yield and quality by using recombinant DNA technology. The other object of the invention is to produce human interferon gamma in active form which allows correct folding in vivo. Yet another object of the invention is to provide a purification scheme with least degradation or aggregation of the molecules of human interferon gamma so produced and recovery of the interferon gamma in high purity and activity.

The invention establishes conditions at which proteins are synthesized in Escherichia coli cells and fold in vivo into the correct conformation with improved yield and quality (homogenous structure) of the protein with very low antigenicity. Lower expression rates induced by a combination of a low incubation temperature and inducer concentration besides other operational parameters in the fermenter allow a correct in vivo folding of the protein. More than 15% of the interferon gamma remains in soluble/free-form in the cytoplasm of the bacterial cell. The advantage of this system is a higher yield after purification and the recovery of active, structurally homogeneous population of the protein.

The reduction of the expression temperature in combination with a reduction of inducer concentration leads to "free-form' of recombinant human interferon gamma that can be easily purified from the cytosolic fraction without any degradation of the specific protein. This protocol omits the guanidium hydrochloride or urea based denaturation/renaturation process and leads to recombinant, active interferon gamma in pure form comparable to the inclusion body purification scheme.

There are numerous methods to accomplish purification of recombinant human interferon gamma. Those methods, which can accomplish the separation under the mildest conditions, to minimize the degradation of mterferon gamma, are the most desirable. This invention has found that the following sequence of purification steps is most suitable for the purpose : i) Primary clarification ii) Centrifugation in) Homogenization iv) Centrifugation v) Cation Exchange Chromatography vi) Dialysis or Gel-Filtration Chromatography

vii) Absolute filtration (0.22 μm using low protein binding

membrane) Accordingly, the present invention provides for a process for producing purified, biologically active, 'free form' of recombinant human interferon gamma (HIG) by cytoplasmic expression in Escherichia coli (E. coli) as bacterial host, comprising the steps of : a) Growing a culture of E. coli inoculated with a plasmid carrying the gene of HIG in a fermenter at a temperature of 22°C to 30°C, under aeration at 0.2 to 1.0 wm, agitation at 200 to 700 rpm, pH 6.5 to 7.5 and dissolved oxygen (DO) 40 to 60%; b) Inducing the culture at an OD (optical density @ 600 nm) 5.0 with an inducer selected from the group consisting of IPTG (Isopropyl - b - D - tbiogalactopyranoside), IAA (Indole Acrylic Acid), IPA (Indole propionic acid) and Lactose at a concentration of 0,005% to 0.05%; c) Harvesting the fermentation broth 5 hours after induction; d) Inactivating culture in the harvest broth with benzyl alcohol; e) Concentrating the inactivated harvest broth by primary clarification through a microfilter; f) Disrupting the concentrated inactivated cells in harvest broth by homogenization at 600 bar in three passes; g) Centrifuging the final homogenate; h) Purifying the 'free form' of recombinant human interferon gamma from the supernatant of step (g) by cation exchange chromatography; and i) Recovering the purified recombinant human interferon gamma after a final step of dialysis/Gel-filtration chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1: Bioprocess flow diagram of interferon gamma purification process of the present invention.

Fig 2: Bioprocess flow diagram of a preferred embodiment of the present invention. Fig 3: Shows the results of SDS-PAGE (Sodium Dodecyl Sulphate -

Polyacrylamide Gel Electrophoresis) of total homogenate, supernatant, cation exchange fraction and the final purified recombinant human Interferon Gamma after gel filtration (photo).

DETAHJED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fully understood, the following detailed description is set forth.

The present invention provides a process for the biosynthesis and purification of the recombinant human interferon gamma. In the specific embodiment of this invention interferon gamma is produced at a lower temperature and inducer concentration in Escherichia coli cells transformed with a plasmid carrying the gene for interferon gamma, grown in a culture, and expressing the gene in the form of interferon gamma protein. Such cells generally show a degree of expression of interferon gamma ranging from 10 to 40% of total cellular protein content. This controlled expression leads to the formation of recombinant human interferon gamma in "free form' within the cells. The production of recombinant human interferon gamma using Escherichia coli is carried in batches ranging from volume of 100 ml to 5000 ml. After fermentation, the interferon containing Escherichia coli cells are recovered from the broth for isolation and purification. The following is the description for the fermentation and cell recovery process. Preparation and maintenance of stock cultures

A stock culture is prepared jn sterile culture flasks containing 10 to 50 ml of sterile Luria Broth medium.

This medium is then inoculated with primary culture of Escherichia coli. The inoculated flask is then incubated on a shaker at 25°C to 37°C, until the absorbance at 600 nm reaches approximately 0.6 to 1.0 OD. This culture is added to a glycerol mixture in 1:1 ratio.

100 μl aliquots of this is dispensed in sterile vials and stored at -70 °C. Each fermentation

batch is started with a replicate stock culture for inoculum. Inoculum preparation

The inoculum is prepared in sterile medium having the following composition :

Components %

K₂HPO₄ 0.5 - 0.8 (NH₄)₂SO₄ 0.1 -0.3

Fe SO₄.7H₂O 0.02 - 0.07

Casamino acids 0.2 - 0.8

MgSO₄ 0.01 - 0.03

CaCl₂ 0.5 - 1.5 The inoculum is incubated in shake flasks at about 37°C for approximately 8 to 10 hours. The inoculum is transferred to a fermentor. The volume of the inoculum is between 2 to 10% of the volume of the fermentor. Fermentation :

Recombinant human interferon gamma fermentation is carried out in fermentors with

working volume of about 5-7.5 litres. The fermentation medium is composed of

Components %

K₂HPO₄ 0.30-1.00

KH₂PO₄ 0.10-0.60

(NH₄)₂SO₄ 0.10-0.30

FeSO₄.7H₂O 0.02-0.05

Casamino acids 0.10 - 0.80

MgSO₄ 0.00-0.01

CaCΪ₂ 0.50-1.50

Glycerol (C₃HsO₃) 0.20 - 0.80

Ampicillin (C₆H₁₈N₃O₄SNa) ^' 3.00-8.00

J TG(C₉H₁₈O5S) 0.005-0.05

Dextrose 30.00 - 80.00

ThiamineHCl(Ci₂H₁₇CIN₄OS.HCl) 0.00-0.005

Ingredients in the medium are sterilized by heat treatment or filtration prior to use in

fermentation. The fermentation is carried out at 22 °C to 30°C. Other operating conditions are as follows:

i) agitation.(rpm) 200 to 700

ϋ) aeration (wm) 0.2 to 1.0 (supplemented with O₂ when necessary)

Hi) pH 6.5 to 7.5 (controlled by addition of acid & base) iv) DO 40 to 60%

Apart from IPTG (Isopropyl-b-D-thiogalactopyranoside), the other inducers which can be used includes IAA (Indole Acrylic Acid), IPA (Indole Propionic Acid), Lactose, etc. Purification :

The cells are inactivated by one of the standard methods e.g. by addition of a chemical killing agent such as 0.25% benzyl alcohol. Primary clarification/concentration of the harvested fermentation broth was done by

microfiltration on Pellicon membrane (Millipore Co) with a cutoff of 0.22 μm. The

concentrated Escherichia coli cells are in a medium containing salts in appropriate buffer (after the buffer exchange in the primary clarification step) in the pH range of 6.0 to 9.0 preferably 8. Recombinant human interferon gamma is extracted by cell disruption of the cell suspension in a high-pressure homogenizer such as APN Gaulin-30 CD. The homogenization is carried out in three passes of 600 bar each. The temperature of the homogenate was maintained below 25 °C by chilled jacket. Then the final homogenate is centrifuged using the Beckman Avanti J 30-1 centrifuge at 20,000 g for 20 minutes at 4°C. The resulting supernatant contains the 'free form' interferon gamma to the concentration of 15% and above. Thus obtained solution of interferon gamma is loaded on a column with source 30S (Amersham Pharmacia) equilibrated in 20 mM sodium phosphate buffer of pH 8.0. Loading is effected at a rate of 1.0 to 4.0 ml min. The column is washed out with 5 column volumes of equilibration buffer (20 mM sodium phosphate buffer of pH 8.0). Then the elution is carried in the following sequence: a) a linear gradient with buffer (0.0 to 0.4 M ΝaCl, 20 inM sodium phosphate buffer, pH 8.0), 3 column volumes at 2-4 ml min. b) step gradient with buffer (0.4 M ΝaCl, 20 mM sodium phosphate buffer, pH 8.0), 2 column volumes at flow rate of 2-4 ml/min, and c) a linear gradient with buffer (0.4 M to 0.8 M ΝaCl, 20 njM sodium phosphate buffer, pH 8.0) five column volumes, at 2-4 ml min flow rate. Finally, interferon gamma is eluted at 0.4 to 0.6 M NaCl concentration in the linear gradient step (steps).

The eluants off the column are monitored by SDS-PAGE and the fractions containing pure interferon gamma are pooled for dialysis (against 2-10 mM succinic acid with 0.9% NaCl at pH of 5.0 to 5,5 for 36 to 50 hours at 4°C to 8 °C or gel-filtration using SG-75 ® matrix (Amersham Pharmacia).

The dialyzed solution is then centrifuged at 20,000 g for 10 minutes. The resulting supernatant is then diluted with the same buffer as used for SDS-PAGE monitoring to a final protein concentration of 0.4 to 0.8 mg/ml. The solution of purified interferon gamma is

passed through 0.22 μm low protein binding absolute filter and stored at -20 °C or below.

The procedure results in a pure, biologically active recombinant human interferon gamma. The novel procedure has consistently produced interferon gamma, having purity above 95% and yield in excess of 5% of total cell protein. This purified, homogeneous recombinant human interferon gamma produced according to the invention may be utilized in the therapeutic treatment of viral infection, tumor or cancer as well as in immuno modulation application methods.

While some of the preferred embodiments of this invention are presented hereinabove, it is apparent that the basic construction can be altered to provide other embodiments, which utilizes the process of this invention. Therefore, this invention is to be construed without limitation to such specific embodiments, which have been presented hereinabove only by the

way of illustration.

Claims

I CLAIM ;

1. A process for producing purified, biologically active, 'free form' of recombinant human interferon gamma (HIG) by cytoplasmic expression in Escherichia coli (E. coli) as bacterial host comprising the steps of : a) Growing a culture of E. coli inoculated with a plasmid carrying the gene of HIG in a fermenter at a temperature of 22°C to 30°C, under aeration at 0.2 to 1.0 wm, agitation at 200 to 700 rpm, pH 6.5 to 7.5 and dissolved oxygen (DO) 40 to 60%; b) Inducing the culture at an OD (optical density @ 600 nm) 5.0 with an inducer selected from the group consisting of IPTG (Isopropyl - b - D - thiogalactopyranoside), IAA (Indole Acrylic Acid), IPA (Indole propionic acid) and Lactose at a concentration of 0.005% to 0.05%; c) Harvesting the fermentation broth 5 hours after induction; d) Inactivating culture in the harvest broth with benzyl alcohol; e) Concentrating the inactivated harvest broth by primary clarification through a microfilter;

f) Disrupting the concentrated inactivated cells in harvest broth by homogenization at 600 bar in three passes; g) Centrifuging the final homogenate; h) Purifying the 'free form' of recombinant human interferon gamma from the supernatant of step (g) by cation exchange chromatography; and i) Recovering the purified recombinant human interferon gamma after a final step of dialysis/Gel-filtration chromatography.

2. The process as claimed in claim 1 wherein I PTG is used as an inducer in step (b).

3. The process as claimed in claim 2 wherein the inducer concentration is 0.025%.

4. The process as claimed in any of the claims 1, 2 or 3 wherein the temperature in the fermentor is maintained at 26.5°C.

5. The process as claimed in claim 4 wherein the cell inactivation in step (d) is carried out with 0.25% benzyl alcohol.

6. The process as claimed in claim 5 wherein homogenization in step (f) is carried out by using APN Gaulin 30 CD homogenizer.

7. The process as claimed in claim 6 wherein a 0.22 μm PNDF membrane cassette (Millipore) is used as microfilter in step (e).

8. The process as claimed in claim 7 wherein the cation exchange chromatography of step(h) is carried out by elution with a step and linear gradient of ΝaCl in an appropriate buffer.

9. The process as claimed in claim 8 wherein cation exchange resin used for the chromatography is source 30S (Amersham Pharmacia).

10. The process as claimed in claims 9 wherein the cation exchange column used for chromatography in step (h) is loaded with source 30S (Amersham Pharmacia) equilibrated in

20 mM sodium phosphate buffer having a pH of 8.0.

11. The process as claimed in claim 10 wherein said loading is effected at a rate of 1.0 to 4.0 ml/min.

12. The process as claimed in claim 11 wherein cation exchange chromatography is carried out by elution with a step and linear gradient of ΝaCl in an appropriate buffer to maintain pH of 8.0.

13. The process as claimed in claim 1 wherein said dialysis is carried out against 2-10 mM succinic acid with 0.9% ΝaCl at pH 5.0 to 5.5 for 36 to 50 hours at 4°C to 8°C.

14. The process as claimed in claim 1 wherein gel filtration is carried out by using SG-75 matrix (pharmacia).

15. The process as claimed in claim 1, wherein centrifugation of final homogenate in step (g) is carried out at 20,000 g for 20 minutes at 4°C.

16. The process for producing purified biologically active "free form" of recombinant human interferon gamma substantially as herein described particularly with reference to the examples.