Cloning, expression and purification method for the preparation of ranibizumab

Abstract

A polynucleotide sequence and a method for Ranibizumab cloning, expression and production having better yield and biologically active protein.

Claims

1. A process for the preparation of Ranibizumab comprising steps of: a) transforming a first host cell with a first vector comprising a first polynucleotide encoding a signal sequence of SEQ ID NO: 5 operably linked to a light chain of Ranibizumab of SEQ ID NO: 2, wherein the first polynucleotide is operably linked to an inducible promoter system; b) transforming a second host cell with a second vector comprising a second polynucleotide encoding a signal sequence of SEQ ID NO: 6 operably linked to a heavy chain of Ranibizumab of SEQ ID NO: 4, for heavy chain of wherein the second polynucleotide is operably linked to an inducible promoter system; c) separately culturing the first host cell and the second host cell in a growth medium; d) expressing the light chain of Ranibizumab in the first host cell and the heavy chain of Ranibizumab in the second host cell as periplasmic inclusion bodies; e) solubilizing the inclusion bodies; and f) refolding in-vitro the solubilized light chain and heavy chain of Ranibizumab.

2. The process of claim 1, wherein the signal sequence of SEQ ID NO: 5 in the expressing step directs the first host cell to transport the light chain of Ranibizumab to a periplasmic space of the first host cell.

3. The process of claim 1, wherein the signal sequence of SEQ ID NO: 6 in the expressing step directs the second host cell to transport the heavy chain of Ranibizumab to a periplasmic space of the second host cell.

4. The process of claim 1, wherein the first host cell in the culturing step is cultured to an OD600 of about 50.

5. The process of claim 1, wherein the second host cell in the culturing step is cultured to an OD600 of about 100.

6. The process of claim 1, wherein the first polynucleotide is nt 8 to nt 718 of SEQ ID NO: 1.

7. The process of claim 1, wherein the second polynucleotide is nt 9 to nt 770 of SEQ ID NO: 3.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 represents expression plasmid carrying Ranibizumab light chain gene construct

(2) FIG. 2 represents expression plasmid carrying Ranibizumab heavy chain gene construct

(3) FIG. 3 represents characterization of recombinant protein expressed using above process

(4) FIG. 4a represents CD Spectra of properly folded Ranibizumab compared with Lucentis

(5) FIG. 4b represents fluorescence Spectra of properly folded Ranibizumab compared with Lucentis

(6) FIG. 5 represents cell based assay showing comparative potency of recombinant Ranibizumab compared with Lucentis

DETAILED DESCRIPTION OF THE INVENTION

(7) Ranibizumab is an affinity maturated Fab fragment derived from bevacizumab. Ranibizumab has a higher affinity for VEGF and also is smaller in size, allowing it to better penetrate the retina, and thus treat the ocular neovascularization associated with AMD (Lien and Lowman, In: Chemajovsky, 2008, Therapeutic Antibodies. Handbook of Experimental Pharmacology 181, Springer-Verlag, Berlin Heidelberg 131-150). Ranibizumab was developed and is marketed by Genentech under the trade name Lucentis.

(8) LUCENTIS is a sterile, colorless to pale yellow solution in a single-use glass vial. Ranibizumab, which lacks an Fc region, has a molecular weight of approximately 48 kilodaltons and is produced by an E. coli expression system in a nutrient medium containing the antibiotic tetracycline. Tetracycline is not detectable in the final product.

(9) Unless indicated otherwise, the term VEGF-binding molecule includes anti-VEGF antibodies, anti-VEGF antibody fragments, anti-VEGF antibody-like molecules and conjugates with any of these. Antibodies include, but are not limited to, monoclonal and chimerized monoclonal antibodies. The term antibody encompasses complete immunoglobulins, like monoclonal antibodies produced by recombinant expression in host cells, as well as VEGF-binding antibody fragments or antibody-like molecules, including single-chain antibodies and linear antibodies, so-called SMIPs (Small Modular Immunopharmaceuticals), as e.g. described in WO002/056910. Anti-VEGF antibody-like molecules include immunoglobulin single variable domains, as defined herein. Other examples for antibody-like molecules are immunoglobulin super family antibodies (IgSF), or CDR-grafted molecules.

(10) VEGF-binding molecule refers to both monovalent VEGF-binding molecules (i.e. molecules that bind to one epitope of VEGF) as well as to bi- or multivalent binding molecules (i.e. binding molecules that bind to more than one epitope, e.g. biparatopic molecules as defined hereinbelow).

(11) Signal sequence refers to a sequence present on the N-terminal side of a secretory protein precursor but absent in the naturally-occurring mature protein, and a signal peptide refers to a peptide cleaved from such a protein precursor. In general, a signal sequence is cleaved by a protease (typically referred to as a signal peptidase) when secreted extracellularly. Although such signal peptides have constant, common features in their sequences among biological species, a signal peptide which exhibits a secretory function in a certain biological species does not necessarily exhibit a secretory function in another biological species.

(12) Inclusion bodies are dense electron-refractile particles of aggregated protein found in both the cytoplasmic and periplasmic spaces of E. coli during high-level expression of heterologous protein. It is generally assumed that high level expression of non-native protein (higher than 2% of cellular protein) and highly hydrophobic protein is more prone to lead to accumulation as inclusion bodies in E. coli. In the case of proteins having disulfide bonds, formation of protein aggregates as inclusion bodies is anticipated since the reducing environment of bacterial cytosol inhibits the formation of disulfide bonds.

(13) Inclusion bodies have higher density (?1.3 mg ml-1) than many of the cellular components, and thus can be easily separated by high-speed centrifugation after cell disruption. Expression of recombinant proteins as inclusion bodies in bacteria is one of the most efficient ways to produce cloned proteins, as long as the inclusion body protein can be successfully refolded. Aggregation is the leading cause of decreased refolding yields.

(14) Protein refolding refers to the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil. Each protein exists as an unfolded polypeptide or random coil when translated from a sequence of mRNA to a linear chain of amino acids. This polypeptide lacks any stable (long-lasting) three-dimensional structure. Amino acids interact with each other to produce a well-defined three-dimensional structure, the folded protein, known as the native state. The resulting three-dimensional structure is determined by the amino acid sequence. Insoluble, inactive inclusion bodies are frequently formed upon recombinant protein production in transformed microorganisms. These inclusion bodies, which contain the recombinant protein in an highly enriched form, can be isolated by solid/liquid separation. After solubilization, native proteins can be generated from the inactive material by using in vitro folding techniques.

(15) The present invention provides new refolding procedure for Ranibizumab comprising efficient in vitro reconstitution of complex hydrophobic, multidomain, oligomeric, or highly disulfide-bonded proteins. These protocols take into account process parameters such as protein concentration, catalysis of disuffide bond formation, temperature, pH, and ionic strength, as well as specific solvent ingredients that reduce unproductive side reactions.

(16) Clone refers to a DNA sequence, such as a gene, that is transferred from one organism to another and replicated by genetic engineering techniques. The present invention comprises genes of heavy and light chain of Ranibizumab with desired modifications for cloning on both the ends. The gene sequences are optimized for better protein expression in E. coli. The synthetic constructs are made so as to have a bacterial leader signal sequence at the N-terminal followed by sequences of gene of interest and two translation stop codons in the end. These signal sequences are selected on the basis of their ability so that they can transport maximum protein in the periplasm of the cell. The signal sequence chosen was taken from a natural bacterial gene.

(17) The main embodiment of the present invention is to use novel cloning processes followed by novel protein expression and its purification procedures for rapid and efficient recovery of recombinant Ranibizumab.

(18) It is yet another embodiment of the present invention to provide novel cloning process of Ranibizumab which comprises transforming the host cell with: i) the vector comprising nucleic acid sequence of SEQ ID No. 1 encoding for light chain of Ranibizumab having an amino acid sequence as shown in SEQ ID No. 2 wherein N-terminal of the said SEQ ID No. 1 is operably linked to a unique signal sequence, start codon and an inducible promoter system ii) transforming another host cell with another plasmid vector comprising nucleic acid sequence of SEQ ID No. 3 encoding for heavy chain of Ranibizumab having an amino acid sequence as shown in SEQ ID No. 4 wherein N-terminal of the said SEQ ID No. 3 is operably linked to a unique signal sequence, start codon and an inducible promoter system.

(19) Another embodiment of the present invention is to provide novel protein expression method for the preparation of Ranibizumab which comprises steps of: i) expressing light & heavy chain separately in two different expression host cells ii) exporting protein to the periplasmic space of the cells with the help of a unique signal sequence iii) partially pure light and heavy chain proteins are refolded together in-vitro.

(20) Another embodiment of the present invention is to provide novel protein purification method of Ranibizumab having an amino acid sequence of SEQ. ID. No. 2 and 4, which comprises: i) high cell density culturing of the host cells in a growth medium by maintaining specific culture conditions ii) expression of the protein in the form of periplasmic inclusion bodies iii) protein refolding of both chains together in-vitro iv) purification of correctly folded protein

(21) Yet another embodiment of the present invention is to provide improved purification process of Ranibizumab which comprises: i) in-vitro refolding of protein ii) performing anion exchange chromatography to separate out closely related misfolded protein species iii) performing cation exchange Chromatography followed by iv) ultra filtration/Diafiltration

(22) Yet another embodiment of the present invention is to provide improved purification process of Ranibizumab which is capable to separate out even closely related misfolded protein species (>95% pure protein).

(23) Yet another embodiment of the invention is to provide a nucleic acid sequence of SEQ ID No. 1 which encodes for light chain of Ranibizumab wherein N-terminal of the said SEQ ID No. 1 is operably linked to a unique signal sequence (SEQ ID No. 5).

(24) Yet another embodiment of the invention is to provide a nucleic acid sequence of SEQ ID No. 3 which encodes for heavy chain of Ranibizumab wherein N-terminal of the said SEQ ID No. 3 is operably linked to a unique signal sequence (SEQ ID No. 6).

(25) Yet another embodiment of the invention is to provide use of unique signal sequence containing the amino acid sequence of SEQ ID No. 5 for production of anti-VEGF antibody.

(26) Yet another embodiment of the invention is to provide use of a unique signal sequence containing the amino acid sequence of SEQ ID No. 6 for production of anti-VEGF antibody.

(27) Yet another embodiment of the invention is to provide use of a unique signal sequence containing the amino acid sequence of SEQ ID No. 5 for production of light chain of Ranibizumab.

(28) Yet another embodiment of the invention is to provide use of a unique signal sequence containing the amino acid sequence of SEQ ID No. 6 for production of heavy chain of Ranibizumab.

(29) TABLE-US-00001 LightChainNucleotideSequence (SEQIDNO:1) 5 GAGCTCCATGGAGTTTTTCAAAAAGACGGCACTTGCCGCACTGGTT ATGGGTTTTAGTGGTCCAGCATTGGCCGATATCCAGCTGACCCAGAGCC CGAGCAGCCTGAGCGCAAGCGTTGGTGATCGTGTTACCATTACCTGTAG CGCAAGCCAGGATATTAGCAATTATCTGAATTGGTATCAGGAGAAACCG GGTAAAGCACCGAAAGTTCTGATTTATTTTACCAGGAGCCTGCATAGCG GTGTTCCGAGCCGTTTTAGCGGTAGCGGTAGTGGCACCGATTTTACCCT GACCATTAGCAGCCTGCAGCCGGAAGATTTTGCAACCTATTATTGTCAG CAGTATAGCACCGTTCCGTGGACCTTTGGTCAGGGCACCAAAGTTGAAA TTAAACGTACCGTTGCAGCACCGAGCGTTTTTATTTTTCCGCCTAGTGA TGAACAGCTGAAAAGCGGCACCGCAAGCGTTGTTTGTCTGCTGAATAAT TTTTATCCGCGTGAAGCAAAAGTGCAGTGGAAAGTTGATAATGCACTGC AGAGCGGTAATAGCCAAGAAAGCGTTACCGAACAGGATAGCAAAGATAG CACCTATAGCCTGAGCAGCACCCTGACCCTGAGCAAAGCAGATTATGAA AAACACAAAGTGTATGCCTGCGAAGTTACCCATCAGGGTCTGAGCAGTC CGGTTACCAAAAGTTTTAATCGTGGCGAATGCTAATAGAAGCTTGGTA CC3 LightChainAminoacidSequence (SEQIDNO:2) DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIY FTSSLIISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC HeavyChainNucleotideSequence (SEQIDNO:3) 5 GAGCTCATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCT GCTCCTCGCTGCCCAGCCGGCGATGGCCGAAGTTCAGCTGGTTGAAAGC GGTGGTGGTCTGGTTCAGCCTGGTGGTAGCCTGCGTCTGAGCTGTGCAG CAAGCGGTTATGATTTTACCCATTATGGTATGAATTGGGTTCGTCAGGC ACCGGGTAAAGGTCTGGAATGGGTTGGTTGGATTAATACCTATACCGGT GAACCGACCTATGCAGCAGATTTTAAACGTCGTTTTACCTTTAGCCTGG ATACCAGCAAAAGCACCGCATATCTGCAGATGAATAGCCTGCGTGCAGA AGATACCGCAGTTTATTATTGTGCCAAATATCCGTATTACTATGGCACC AGCCACTGGTATTTCGATGTTTGGGGTCAGGGCACCCTGGTTACCGTTA GCAGCGCAAGCACCAAAGGTCCGAGCGTTTTTCCGCTGGCACCGAGCAG CAAAAGTACCAGCGGTGGCACAGCAGCACTGGGTTGTCTGGTTAAAGAT TATTTTCCGGAACCGGTTACCGTGAGCTGGAATAGCGGTGCACTGACCA GCGGTGTTCATACCTTTCCGGCAGTTCTGCAGAGCAGCGGTCTGTATAG CCTGAGCAGCGTTGTTACCGTTCCGAGCAGCAGCCTGGGCACCCAGACC TATATTTGTAATGTTAATCATAAACCGAGCAATACCAAAGTGGATAAAA AAGTTGAGCCGAAAAGCTGCGATAAAACCCATCTGTAATAGGGTACC3 HeavyChainAminoacidSequence (SEQIDNO:4) EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGNINWVRQAPGKGLEWV GWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMINSLRAEDTAVYYC AKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTVICNVNHKPSNTKVDKKVEPKSCDKTHL

(30) The amino acid sequence of the unique signal sequence for light chain of Ranibizumab is:

(31) TABLE-US-00002 (SEQIDNo:5) MEFFKKTALAALVMGFSGAALA

(32) The amino acid sequence of the unique signal sequence for heavy chain of Ranibizumab is:

(33) TABLE-US-00003 (SEQIDNo:6) MKYLLPTAAAGLLLLAAQPAMA

(34) The invention will now be further described by the following examples, which are illustrative rather than limiting.

Example 1

Generation of pCDL18-LC Vector for Light Chain of Ranibizumab

(35) Bacterial expression vector for light chain of Ranibizumab was generated by cloning light chain of Ranibizumab along with a signal sequence (SEQ ID No: 1) at the 5 end into the NcoI/HindIII site in multiple cloning site (MCS) of pCDL18-LC vector.

(36) Key components of synthetic gene cassette and schematic design are as given below.

(37) ##STR00001##

(38) Generation of pCDL18-HC Vector for Heavy Chain of Ranibizumab

(39) Bacterial expression vector for heavy chain of Ranibizumab was generated by cloning heavy chain of Ranibizumab along with a signal sequence (SEQ ID No: 3) at the 5 end into the NdeI/XhoI site in pCDL18-HC vector.

(40) Key components of synthetic gene cassette and schematic design are as given below.

(41) ##STR00002##

Example 2

Transformation of Light Chain Gene in BL21 (DE3)

(42) pSR04 vector carrying expression construct for light chain gene (pCDL18-LC) was transformed in BL21 (DE3) and recombinant clones were selected. The transformants were plated on LB agar plates containing kanamycin (30 ?g/ml) for selection. Protein expression analysis was also performed after inducing the cells with 1 mM IPTG for 4 h in a shake flask. Whole cell lysate and extracellular protein samples were analyzed for clone selection.

(43) Transformation of Heavy Chain Gene in BL21 (DE3)

(44) pSR02 vector carrying expression construct for heavy chain gene (pCDL18-HC) was transformed in BL21 (DE3) and recombinant clones were selected. The transformants were plated on LB agar plates containing ampicillin for selection. Protein expression analysis was also performed after inducing the cells with 1 mM IPTG for 4 h in a shake flask. Whole cell lysate and extracellular protein samples were analyzed for clone selection.

Example 3

General Expression of Light & Heavy Chain Separately in Two Different Expression Host Cells in E. coli

(45) To maximize the desired protein expression the light and heavy chains of Ranibizumab were cloned into two separate vector systems and transformed individually in two different E. coli cells. The protein expression is derived from T7 promoter system. Both these constructs are carried in by these high copy number plasmids and capable of expressing protein in a tightly regulated manner.

(46) The overexpressed recombinant proteins are purified and characterized. The first 10 residues at the N-terminal were confirmed to be DIQLTQSPSS (aa 1 to 10 of SEQ ID NO: 2) for light chain and EVQLVESGGG (aa 1 to 10 of SEQ ID NO: 4) for the heavy chain, which is the authentic start sequence of both the chains. A clear and unambiguous signal was obtained for all the 10 residues.

Example 4

Generation of Targeted Protein in Form of Periplasm Inclusion Bodies

(47) The recombinant E. coli cells were cultivated in the shake flasks (seed flasks) for inoculum preparation and for production, the inoculum obtained from seed flask was transferred to production fermenter and cultured for 25 h in fed batch mode. During fermentation, the cells were provided with air and oxygen by means of sparging. The growth of the cells was maintained by controlled addition of feed (Nutrient supplements) in pH stat mode, the pH was maintained by supplying glucose and nitrogen in feed to reduce the pH of the batch. Base (NaOH) was used to increase the pH of the batch as needed. Cells were induced with IPTG at 20th hour of batch age and the fermentation is carried out for another 5 hours. The targeted protein is produced in the form of periplasmic IBs.

(48) For light chain the cell culture density (OD600) at harvest was ?50 and biomass obtained from that was around 100 g/L, which yielded around 25 g IBs/L.

(49) Similarly for heavy chain the cell culture density (OD600) at harvest was ?100 and biomass obtained was around 155 g/L which yielded around 33 g IBs/L.

Example 5

Refolding of Light and Heavy Chain Together

(50) 25 g of LC and HC each were solubilized separately in 500 mL of solubilization buffer containing 6 M GuHCl and pooled in 1:1 ratio. Pooled SIB was reduced with 4 mM of DTT for 1 h. Reduced LC and HC pool was subjected to oxidation with 10 mM cystine and incubated for 3 h. Reduced and oxidized SIB were diluted 25 times in the refolding buffer (100 mM Tris, 0.6 M Arginine, 5% Sorbitol, 2 mM EDTA, pH 9.0) by slow addition. The 0.6 mM of cystine and 0.75 mM of cysteine was added to the refolding mixture and reaction was incubated at (2-8)? C. for ?5 days Refolding output was concentrated using 10 kDa membrane and diafiltered against 50 mM Tris buffer, pH 9.0.

(51) Yield and purity after refolding: Properly folded Ranibizumab yield after refolding was around 18% of the total protein.

Example 6

Purification for Properly Folded Protein

(52) Refolding output was concentrated using 10 kDa membrane and diafiltered against 50 mM Tris buffer pH 9.0. Diafiltration output was loaded on Q Sepharose FF resin in binding mode and protein was eluted out by reducing the pH to 6.7 in linear gradient in 10 CV from 0% B to 100% B. Q Sepharose Output was loaded on SP Sepharose HP resin at pH 5.0. Protein was eluted out by increasing salt concentration as follows 20% step followed by 20% to 50% gradient and finally a 100% step gradient. Pooled fractions were concentrated and diafiltered against the formulation buffer using a 10 kDa membrane.

(53) Yield of final product after purification: The overall protein purification process recovery was around 9% with a purity level of >99%.