EP1833846A2 - Method for production of a therapeutically pure granulocyte macrophage - colony stimulating factor - Google Patents
Method for production of a therapeutically pure granulocyte macrophage - colony stimulating factorInfo
- Publication number
- EP1833846A2 EP1833846A2 EP05732861A EP05732861A EP1833846A2 EP 1833846 A2 EP1833846 A2 EP 1833846A2 EP 05732861 A EP05732861 A EP 05732861A EP 05732861 A EP05732861 A EP 05732861A EP 1833846 A2 EP1833846 A2 EP 1833846A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- csf
- processed
- protein
- unprocessed
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
- C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
Definitions
- the present invention relates to a method for production of a therapeutically pure granulocyte macrophage-colony stimulating factor (GM-CSF).
- GM-CSF granulocyte macrophage-colony stimulating factor
- the instant invention relates to the production of a recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF with a heterologous PeIB signal peptide in Escherichia coli (E.coli), processing of the pre-GM-CSF form by the bacterial host by cleaving the signal sequence and extracting the processed form from the membrane pellet fraction of the cell lysate for final purification.
- GM-CSF granulocyte macrophage-colony stimulating factor
- E.coli Escherichia coli
- the invention is directed to a recombinant E.coli strain harboring a vector construct comprising the mature GM-CSF coding sequence fused to the PeIB signal peptide and a simplified process for the purification of an essentially endotoxin free, processed GM-CSF protein for therapeutic applications from a periplasmic but insoluble cellular localization.
- Human GM-CSF belongs to the family of endogenous colony stimulating factors (CSFs) that stimulate the growth of hematopoietic progenitor cells of the myeloid and erythroid lineages. It is a naturally occurring hematopoietic growth factor (cytokine) that is produced by T cells, macrophages, fibroblasts and endothelial cells. As the name indicates, it was originally defined by its ability to stimulate the proliferation and differentiation of granulocyte/macrophage progenitor cells, but later found to have the same activity on progenitor cells of other hematopoietic lineages (Metcalf D.
- CSFs colony stimulating factors
- CSFs colony-stimulating factors
- GM- CSF "turns on” the immune system by stimulating production of the myeloid progenitor stem cells of neutrophils, monocytes, macrophages, eosinophils, and dendritic cells. It also has a role in functional cell activities, such as T cell activation. Increased monocyte activity generates macrophages and allows for increased phagocytosis that may prevent or diminish the occurrence of life- threatening bacterial and fungal infections.
- Endogenous myeloid colony-stimulating factors have demonstrated the ability to enhance the clinical management of immuno- suppressed patients with cancer. These agents are associated with significant decreases in chemotherapy-associated infections, antibiotic use, length of hospital stay and mortality. Clinical trials suggest that GM-CSF has clinical benefits beyond enhancing neutrophil recovery, including shortening the duration of mucositis and diarrhea, stimulating dendritic cells, preventing infection, acting as an adjuvant vaccine agent, and facilitating antitumor activity.
- GM-CSF is being used as a vaccine adjuvant.
- Naturally occurring mature GM-CSF is a glycoprotein containing 127 amino acids and two disulfide bonds.
- the availability of GM-CSF from its natural source is extremely limited because of its presence in trace quantities. This hindered the biochemical characterization of the cytokine until the time when it was expressed using a recombinant expression system.
- GM-CSF The mammalian cell-expressed GM-CSF is present in different glycosylated forms ranging in size from 14 to 35 kilo Daltons. Some forms of GM-CSF may contain two N-linked carbohydrate groups and/or three O-linked carbohydrate groups explaining the apparent size heterogeneity.
- the GM-CSF expressed in CHO cells is biologically active. However, the biological activity is enhanced by 20-fold upon enzymatic removal of the carbohydrate residues. Thus, indicating that the unglycosylated form may be superior for clinical use (Moonen P. J., et al., 1987 (Proc natl Acad Sci USA).
- Human GM-CSF in unglycosylated form has been synthesized in high yield using a temperature inducible plasmid in Escherichia coli (Burgess, A.W., et ah, 1987 (Blood 58: 43-51)).
- The.fi'. co/i-expressed GM-CSF formed inclusion bodies in the cytoplasm and also had an extra methionine residue at the N-terminus.
- GM-CSF Human granulocyte macrophage— colony stimulating factor
- U.S. Patent 5,047,504 (Boone; Thomas C, assigned to Amgen Inc., February 17, 1989) describes recovering GM-CSF from a microorganism in which it is produced by lysing the microorganism, separating insoluble material containing GM-CSF from soluble proteinaceous material, extracting the material with a chaotropic agent, refolding and oxidizing the GM-CSF in the presence of a glutathione, precipitating the incorrectly folded GM-CSF, removing the denaturant and subjecting the residual GM-CSF to ion exchange and reverse phase chromatography to recover purified GM-CSF.
- the U.S. Patent 5,136,024 describes a method for extracting GM-CSF from bacterial cells by treating it with an acid to lower the pH to a value between 1.5 and 3.0 and then neutralizing the suspension to a pH between 6 and 9.
- GM-CSF in this case was cloned with the signal peptide and hence the processed form of GM-CSF without the N-terminal methionine is likely to be obtained after purification but the procedure mentioned is only for extraction by acidification and neutralization and does not mention purification of the protein to homogeneity for therapeutic use.
- U.S. Patent 5,391,706 discusses the purification of GM-CSF from bacteria to 95% homogeneity by a four-step chromatography process that includes QAE, dye-ligand affinity, gel filtration and reverse phase steps.
- a related patent to the same assignee (Schering Corporation), US 5,451,662 describes an ion-exchange separation procedure near the isoelectric point of the protein called delta isoelectric point chromatography. In this patent also no mention is made of the form (processed or unprocessed) of GM-CSF that is purified and its therapeutic quality.
- the present invention provides a method of production of human GM- CSF in a bacterial host by transforming it with a genetically engineered plasmid that contains the gene for human GM-CSF with the heterologus signal peptide, culturing these recombinant microbial cells under favorable conditions for enhanced expression of the protein and purifying it to therapeutic endotoxin- free grade by a simple, scaleable two-step chromatography process that does not include periplasmic extraction or solubilization and refolding from inclusion bodies.
- the present invention provides a method for production of a therapeutically pure granulocyte macrophage - colony stimulating factor (GM-CSF), wherein said GM-CSF from a recombinant microorganism, is secreted in its mature form has no methionine residue at N-terminus, and the method comprises: a) cloning the human GM-CSF gene in- frame with Pel B signal peptide in a bacterial strain b) producing elevated expression levels of the processed protein by altering the induction conditions c) culturing GM-CSF producing recombinant cells in which over-expressed GM-CSF gets processed during secretion into the periplasm and gets associated with the cell membranes.
- GM-CSF granulocyte macrophage - colony stimulating factor
- the membrane pellet fraction containing GM-CSF is recovered from the cells by lysing them by high-pressure homogenization or sonication.
- the membrane pellets can be obtained from the lysate, for example by centrifugation. The pellets are then subjected to a repeated washing procedure to remove endotoxins.
- Such washing is affected by using either or a combination of a non- ionic detergent and a chaotropic agent.
- the processed GM-CSF that is bound to the cell membrane after secretion is then extracted by a combination of a low concentration of a chaotropic agent and any ionic salt.
- the extracted soluble GM-CSF is buffer exchanged and directly loaded onto an anion exchange column for partial purification and enrichment of the collected fraction with GM-CSF.
- the eluate from this column is then purified to homogeneity on a hydrophobic column which when eluted with the buffer gives a homogeneous protein of GM-CSF with a recovery of almost 80% of the processed form that was initially bound to the membrane fraction.
- Figure 1 is a map of E. coli expression vector, which directs the secretion of GM-CSF
- Figure 2 is the nucleotide and amino acid sequence of mature GM-CSF
- Figure 3 is the elution profile of GM-CSF after chromatography- I
- Figure 4 is the elution profile after chromatography-II showing separation of processed and unprocessed GM-CSF
- Figure 5 is the SDS-PAGE analysis of final purified GM-CSF under reducing and non-reducing conditions showing a single processed GM- CSF band and the absence of the unprocessed GM-CSF and other host cell impurities DETAILED DESCRIPTION OF THE INVENTION WITH
- the present invention provides a method for the production of a fully processed form of human GM-CSF by recombinant methods using a microbial expression system with improved culturing conditions and a simple and scaleable purification process for an endotoxin- free preparation of the purified protein.
- the process described in the present invention can be applied to the production of recombinant GM-CSF for therapeutic purposes.
- the mature coding portion of the GM-CSF gene is isolated from a human lymphocyte cell line.
- Total RNA is extracted from the cells and converted into cDNA.
- An aliquot of the synthesized cDNA is used as a template for amplifying the desired DNA fragment of GM-CSF coding sequence using appropriately designed specific oligonucleotide primers.
- the GM-CSF amplicon is cloned at the Bam Hl and blunt fragment into the expression vector in frame with the Pel B signal sequence at its 5 ' end to form the plasmid construct pTMF02 as shown in Fig:l.
- a promoter inducible either by IPTG/ lactose / arabinose will drive the transcription of the Pel B-mature hGM-CSF coding sequence.
- the complete nucleotide sequence of the GM-CSF gene is given in Fig: 2.
- E. coli host strains harboring the afore described plasmid construct will secrete the mature GM-CSF into the periplasmic space when grown using optimized media conditions.
- the optimized culture conditions comprises of induction with an inducer of the promoter like IPTG, arabinose or lactose or related sugar molecules, the presence of a reducing agent like glutathione, DTT, L-cysteine or related compounds having thiol groups to facilitate reshuffling of incorrect disulfide bonds and growth at low temperatures of 20 to 28 0 C for slower growth kinetics.
- an inducer of the promoter like IPTG, arabinose or lactose or related sugar molecules
- a reducing agent like glutathione, DTT, L-cysteine or related compounds having thiol groups
- Cultivation of recombinant E. coli strain containing human GM-CSF gene is done under conditions optimized for maximum heterologous protein expression. Fermentation is carried out in a 10-litre fermentor vessel. The following parameters are maintained during the ran viz., air flow: 1 vessel volume per minute (vvm), agitation: 300-800 rpm, dissolved oxygen (DO): >40% and temperature around 37 0 C during cell growth, which is shifted to 20 - 28 0 C prior to the addition of the inducer and maintained as such till harvest.
- air flow 1 vessel volume per minute (vvm)
- agitation 300-800 rpm
- DO dissolved oxygen
- Production medium used is a complex medium like Terrific Broth, containing glycerol as carbon source, yeast extract and tryptone as nitrogen sources. Ampicillin and chloramphenicol are used for selection.
- the medium is inoculated with a seed culture and allowed to grow till the desired cell density is obtained and the temperature is gradually shifted to between 20 and 28 0 C, for acclimatization, and maintained thereon i.e., during induction and post- induction growth.
- GM-CSF is achieved by feeding of the inducer selected from the family of sugars that can induce the promoter at concentrations ranging from 0.ImM to 1OmM, while a reducing agent is added in a concentration range of 0.ImM to 2OmM to improve the yield of processed GM-CSF.
- the inducer selected from the family of sugars that can induce the promoter at concentrations ranging from 0.ImM to 1OmM
- a reducing agent is added in a concentration range of 0.ImM to 2OmM to improve the yield of processed GM-CSF.
- the batch is harvested.
- the cellular biomass obtained is atleast 40 ⁇ 10 g/1 (wet wt).
- the secreted GM-CSF is directed to the E. coli periplasm by a signal sequence that gets processed during the process. However, a fraction of the GM-CSF molecules remain unprocessed and retain the signal sequence which are separated from the processed form during the protein purification process. Purification
- Purification of GM -CSF from the harvested E. coli cells is done by a simple procedure involving lysis of the cells, solubilization of membrane bound secreted GM-CSF followed by a two-step chromatography procedure.
- the highlight of the simplified.purification process is a selective purification step from the membrane fraction for the recovery of the processed GM-CSF protein from a mixture of the processed and unprocessed GM-CSF protein.
- the unprocessed form containing the signal peptide shows a differential solubility and binding behaviour on the chromatography column.
- the harvested cell pellet is suspended in lysis buffer, for example, 50gms of cell paste is suspended in 500ml to 1 litre of lysis buffer, i.e. 25mM to 5OmM Tris buffer, at pH ⁇ .O, ImM to 1OmM EDTA and 0.5mM to ImM phenyl methyl sulfonyl fluoride (PMSF) and lysed by high pressure homogenization or by repeated short pulses on a sonicator.
- the process is conducted in an ice bath and continued till complete cell lysis is obtained as monitored by the stabilization of OD 600 value of the lysate.
- the cell lysate is centrifuged in a refrigerated highspeed centrifuge to pellet the cell debris along with the membrane fraction.
- This cell pellet is rid of contaminating cell wall lipids and endotoxins by washing the fraction with either a non-ionic or an ionic detergent or a combination of both.
- the non-ionic detergent can be chosen from a class belonging to Triton or Tween series and the ionic from a class belonging to the any of the anionic detergents.
- the pellet thus obtained has the processed and unprocessed GM-CSF bound to the membrane, which can be easily solubilized in native form by treating it with a high molarity salt solution or a mild chaotropic solution or a combination of both.
- the solubilized protein fraction is recovered by centrifugation and buffer exchanged with the equilibration buffer of the first chromatography column step to match the conductivity and pH values with the column loading conditions.
- a chromatography column is packed with an anionic matrix selected from a group of Q, DEAE or QAE ligands. This is equilibrated with a suitable buffer in the pH range of 7.0 to 8.5 and the loaded at low conductivities.
- the solubilized sample is loaded on to the column and washed with equilibration buffer till the optical density value at 280nm returns to baseline.
- GM-CSF is eluted from this column using a linear gradient of salt preferably sodium chloride from 0 M to 1.0 M concentration in the equilibration buffer.
- the processed and unprocessed GM-CSF elutes from this column as a distinct single peak as shown in Fig 3. • Most of the GM-CSF loaded onto the column can be recovered almost quantitatively with this elution step without any significant drop in yield.
- the processed mature form of GM-CSF (without the signal peptide) can be selectively purified to homogeneity from a mixture of processed and unprocessed forms by a second chromatography step on either a reverse phase HPLC column or a hydrophobic interaction chromatography column.
- the protein eluate after the first chromatography step is treated with trifluoroacetic acid in the range of 0.1 to 0.2 % to lower the pH to a value below 3.0 and is loaded onto a butyl, octyl or octadecyl reverse phase HPLC semi-prep column preferably butyl or octyl column equilibrated with a suitable concentration of TFA in water.
- the bound GM-CSF protein is eluted from the column by a linear gradient from 20% to 100% B.
- the processed GM-CSF protein elutes from the column as a single distinct peak.
- the sample can be loaded onto a hydrophobic interaction column having the octyl or butyl functional groups at medium to high conductivity range.
- Both forms of GM-CSF bind to the column under these conditions and the processed form can be selectively eluted by lowering the conductivity of the elution buffer.
- the unprocessed fo ⁇ n being more hydrophobic needs a greater reduction in conductivity to elute form the column. This brings about a clear separation of the processed form from the unprocessed form of GM-CSF as shown in Fig 4.
- the GM-CSF protein obtained after elution from the reverse phase or HIC column is free of the unprocessed fo ⁇ n of GM-CSF and also other E.
- EXAMPLE 1 This shows that high yields of processed GM-CSF are obtainable under conditions where an inducer in combination with a reducing agent at appropriate temperature drives the transcription of the GM-CSF coding sequence with the signal peptide.
- Inducers can be chosen from a family of sugar molecules like IPTG, arabinose or lactose or their analogs at concentration ranges from ImM to lOmM.
- the microbial host strain harboring the plasmid construct secretes mature GM-CSF into the periplasmic space when grown initially at around 37 degrees Centigrade using the optimal media conditions.
- the presence of a reducing agent containing one or more thiol groups in the concentration range of ImM to 2OmM will facilitate reshuffling of incorrect disulfide bonds and hence better secretion of the processed form.
- lower growth temperatures in the range of 20 to 28 degrees Centigrade can also give slower growth kinetics and more efficient refolding.
- a combination of the above parameters may also be chosen to give effective secretion.
- the above parameters can be tried either in isolation or in combination to obtain efficient processing and secretion of GM-CSF suitable as a starting material for production of therapeutic grade of processed GM-CSF.
- This example shows the preparation of membrane pellet after lysis of bacterial cells.
- Bacterial cell pellet after harvesting is suspended in 5OmM Tris HCl buffer pH8.0, 1OmM EDTA, ImM PMSF at a pellet to buffer ratio in the range of 1 :5 to 1 :20, more preferably in the range of 1 : 10 and 1 : 15.
- Lysis of cells is accomplished using a high-pressure homogenizer or sonicator, with multiple passes, keeping the temperature below 4 degrees C and monitoring the OD at 600nm for complete lysis.
- the lysate obtained is centrifuged at high speed 12000 to 16000xg for 15 to 30 minutes at 2 to 8 degrees C to pellet the cell debris and membrane fraction.
- This example relates to the purification of solubilized GM-CSF extracted from the membrane pellet.
- Solubilization is achieved by the addition of high molarity salt solution to the pellet, preferably in the range of 0.5 - 2M NaCl .
- high molarity salt solution preferably in the range of 0.5 - 2M NaCl .
- non-denaturing concentrations of chao tropic agents or detergents can be used either as such or in combination with a salt solution.
- the solubilized fraction containing GM-CSF (having some amount of the unprocessed form of GM-CSF along with the processed GM-CSF) is loaded onto a chromatography column, packed with an anion exchanger selected from a group of various polymer based matrices like cellulose, agarose, dextran or a synthetic polymer based.
- the functional groups can be Q, DEAE or QAE.
- GM-CSF binds to the column in the pH range of 7.0 to 8.5 and can be eluted with good recovery by using a gradient of 0 to IM NaCl in the equilibration buffer.
- the processed protein elutes along with a small contamination of the unprocessed protein which can be eliminated completely in the next chromatography step.
- This example relates to the use of reverse phase HPLC or hydrophobic interaction chromatography for the final purification of processed GM-CSF protein.
- trifluoro acetic acid is added to a final concentration in the range of 0.1 to 0.2 % to lower the pH and is loaded onto a C-4 reverse phase HPLC semi-prep column equilibrated with 0.05% to 0.2% TFA in water.
- the bound GM-CSF protein is eluted from the column by a linear gradient from 20% to 100% B, where B is preferably 0.05% to 0.2%TFA in 70- 95% acetonitrile or isopropanol in water.
- the sample can be loaded onto a hydrophobic interaction column having the octyl or butyl functional groups at medium to high conductivity range in the range of 60 to 15OmS in Tris or phosphate buffer in the pH range of 7.0 to 9.0.
- the high conductivity values can be obtained by the addition of sodium chloride, sodium sulphate or ammonium sulphate salts.
- GM-CSF binds to the column under these conditions and can be selectively eluted by lowering the conductivity of the elution buffer.
- the GM-CSF protein obtained after elution from the reverse phase or HIC column is free of the unprocessed form of GM-CSF and most of the host protein related contaminants.
- the purity level conforms to the standards required for therapeutic proteins with endotoxin units in the range of ⁇ 5EU /mg protein and the host cell DNA and protein limits conforming to the US-FDA specifications of lOOpg / dose equivalent protein and 100ng/mg respectively for these impurities.
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- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
Description
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Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IN1343CH2004 | 2004-12-09 | ||
PCT/IN2005/000088 WO2006061851A2 (en) | 2004-12-09 | 2005-03-18 | Method for production of a therapeutically pure granulocyte macrophage - colony stimulating factor |
Publications (1)
Publication Number | Publication Date |
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EP1833846A2 true EP1833846A2 (en) | 2007-09-19 |
Family
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP05732861A Withdrawn EP1833846A2 (en) | 2004-12-09 | 2005-03-18 | Method for production of a therapeutically pure granulocyte macrophage - colony stimulating factor |
Country Status (3)
Country | Link |
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EP (1) | EP1833846A2 (en) |
AP (1) | AP2007004049A0 (en) |
WO (1) | WO2006061851A2 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2020234742A1 (en) * | 2019-05-20 | 2020-11-26 | Lupin Limited | Granulocyte colony stimulating factor purification |
-
2005
- 2005-03-18 EP EP05732861A patent/EP1833846A2/en not_active Withdrawn
- 2005-03-18 AP AP2007004049A patent/AP2007004049A0/en unknown
- 2005-03-18 WO PCT/IN2005/000088 patent/WO2006061851A2/en active Application Filing
Non-Patent Citations (1)
Title |
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See references of WO2006061851A2 * |
Also Published As
Publication number | Publication date |
---|---|
WO2006061851A3 (en) | 2009-04-23 |
WO2006061851A2 (en) | 2006-06-15 |
AP2007004049A0 (en) | 2007-06-30 |
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