RELATED APPLICATIONS

This application is related to Indian Provisional Application 201721036661 filed 16 ^th Oct, 2017 and is incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to improved method for purification of recombinant PTH (1-34) with decreased protein precipitation or particle formation.

BACKGROUND OF THE INVENTION Parathyroid hormone (PTH) is a polypeptide hormone which is naturally produced in the parathyroid of mammals. The human polypeptide consists of 84 amino acids and is involved in the regulation of the calcium concentration in blood plasma. If the calcium level drops below a threshold level, PTH is secreted by cells of the parathyroid glands into the blood and induces a release of calcium from bone tissue. At the same time, PTH supports calcium absorption from the small intestine and enhances calcium reabsorption from the primary urine, thereby suppressing calcium loss via the kidneys. Due to its calcium releasing effects, an excessive amount of PTH in the blood as commonly observed in primary and secondary hyperparathyroidism has been found to be associated with reduced bone density and bone atrophy (osteoporosis). Considering the physiological effects of PTH, it appears odd that the hormone nevertheless proved useful in the therapy of osteoporosis. However, animal studies in rats for the first time revealed that a short-term exposure to PTH supports bone formation due to transient activation of osteoclasts, whereas a sustained exposure ultimately results in bone atrophy. Subsequent clinical studies in humans using the pharmaceutically active fragment PTH (1-34) of human PTH confirmed that the fragment can be used for treating osteoporosis. PTH (1- 34) is a polypeptide having a molecular mass of 4.7 kDa that consists of the first 34 amino acids of the human PTH hormone. It was approved for osteoporosis therapy in 2002 under the product name "Teriparatide". Teriparatide is sold by Lilly Pharma under the trade name Forteo (in US) and Forsteo (in Europe). It is produced in the form of a fusion protein by recombinant DNA technology using a genetically engineered E. coli strain. The PTH fusion protein expressed resides in the cytoplasm of E. coli in the form of insoluble inclusion bodies (IBs).

Purification of protein is a series of processes intended to isolate a single type of protein from a complex mixture, besides being essential for the characterization of its structure and function. The starting material is usually a biological tissue or a microbial culture expressing PTH (1-34). The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and nonprotein parts of the mixture, and finally separate the desired protein from all other proteins and impurities. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps exploit differences in protein size, physico-chemical properties and binding affinity to variety of chromatographic resins under different conditions.

Because of the importance of recombinant teriparatide in the treatment of osteoporosis, the provision of recombinant teriparatide of high purity and high specific activity is desirable. Recombinant teriparatide requires repeated injections. Highly purified recombinant teriparatide preparations can be administered subcutaneously, permitting self-administration by the patient and thereby increasing patient convenience and compliance.

At present, PTH (1-34) for therapeutic use is produced by heterologous expression in bacterial host cells and subsequent purification of the pharmacological active polypeptide. It is produced in the form of a fusion protein by recombinant DNA technology using a genetically engineered E. coli strain. The PTH fusion protein expressed resides in the cytoplasm of E. coli in the form of insoluble inclusion bodies (IBs). After partial or substantial purification, the fusion protein is treated enzymatically with the enzyme corresponding to the selective cleavage site. Alternatively, the fusion protein in its more impure state, even in refractile form, can be treated with the enzyme. If needed, the resulting mature PTH, or variant thereof, can be further purified. Rathore et al., Journal of Chemical Technology and Biotechnology, 88(10), 1794-1806, 2013 teaches Escherichia coli expression system serves to be the most popular and widely used expression system for production of recombinant therapeutic protein in form of inclusion bodies (IBs). These IBs are dense, with tightly packed aggregated proteins and this aggregated protein are converted into fully active native form by refolding process. Refolding in batch process using drip dilution (of solubilized and reduced IBs) method is the most preferred and widely used. The refolding buffer contains redox components, buffering agent, chelating agents and stabilizers etc.

Post refolding includes several downstream unit operations to purify the recombinant therapeutic proteins. Due to volume handling issues when operated at large scale, the refolding output is concentrated and buffer exchanged to condition it for the next chromatography step.

Callahan et al., Journal of pharmaceutical sciences, 103(3), 862-869, 2014 discloses formation of protein aggregates and observation of millions of micron-sized particles per milliliter in the Ultrafiltration Diafiltration (UFDF) products of multiple proteins prior to final filtration during reduction of the volume and buffer exchange (UFDF) of refolding output. It further, discloses that the complexity of the UF/DF unit operation leads to several hypotheses for the principal mechanism of UF/DF induced particle formation: (1) shear stress or other characteristics of the fluid flow (2) impurities leached into the product stream from contact surfaces during extended periods of recirculation (3) interfacial interactions and (4) pump-specific stresses. Shukla et al., The Journal of Physical Chemistry B, 114(42), 13426-13438, 2010 discloses arginine as the most preferred and extensively used additive in refolding of recombinant proteins. Arginine is widely used to suppress protein aggregation. Arginine reduces attractive protein- protein interactions, and increases the solubility of unfolded species thereby decreasing the rate of association of unfolded and partially folded intermediates on the folding pathway during refolding.

Cromwell et al., The AAPS Journal, 8 (3), E572-E579, 2010 discloses Protein aggregation is a common issue encountered during manufacture of biotherapeutics.

WO2008031020 discloses methods for isolating a product and/or reducing turbidity and/or impurities from a load fluid comprising the product and one or more impurities by passing the load fluid through a medium, followed by at least one wash solution comprising arginine or an arginine derivative and collecting the product using an elution solution.

US 6103495 describes the purification process comprising the following steps (a) separating host cells from the culture medium; (b) subjecting the medium to reverse-phase liquid chromatography and recovering fractions containing peptide product; (c) subjecting said fractions of step (b) to cation exchange chromatography and (d) thereafter recovering fractions containing peptide product.

US6210925 describes the purification process comprising the following steps: (a) separating host cells from the culture medium; (b) subjecting the medium to cation exchange chromatography and recovering fractions containing said peptide product; (c) subjecting the recovered fraction of step (b) to reverse-phase liquid chromatography and recovering fractions containing peptide product; (d) subjecting the recovered fractions of step (c) to cation exchange chromatography and (e) thereafter recovering fractions containing peptide product.

WO2009129226 discloses method for concentrating a protein, in particular a method for concentrating a plasma product, in particular IgG, using glycine in a two-stage ultrafiltration/diafiltration approach.

WO2009019715 discloses two steps orthogonal purification process for recombinant human PTH (1-34) comprising of cation exchange chromatography optionally followed by preparative chromatography selected from HIC or RP-HPLC to yield a target protein of > 99%.

WO2015025335 discloses process for purification of parathyroid hormone using enzymatic cleavage; anion exchange chromatography followed by other suitable purification step.

US20170129935 discloses method for purifying PTH (1-34) by cation exchange chromatography.

Tripathi, ChemBioEng Reviews, 3: 116-133, 2016 discloses refolding process of recombinant proteins produced in E.coli and purification using various chromatographic steps.

In light of the above, new methods are needed which are effective in purifying recombinant PTH (1-34) with reduced precipitation or particle formation. To handle the shear stress and interfacial interactions problem during diafiltration step of the concentrated refolding output, present invention uses critical arginine concentration in the concentrated refolding output solution. The improved method of the present invention provides for the effective purification of the recombinant PTH (1-34) polypeptide by ultrafiltration diafiltration (UFDF) followed by subsequent purification steps. Accordingly, it would be desirable to provide a method for purifying recombinant PTH (1-34) by decreasing protein aggregation/precipitation and particle formation along with increase in optical density to achieve higher final concentrations along with minimizing yield loss and impact on processing time. OBJECT OF THE INVENTION

The principal object of the present invention is to provide an improved process for purification of protein with decreased protein precipitation or particle formation through ultrafiltration diafiltration (UFDF) followed by subsequent purification steps.

Another object of the present invention is to provide an improved process for purifying a protein through ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation followed by subsequent purification steps.

Another object of the present invention is to provide an improved process for purifying recombinant PTH (1-34) by ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation followed by subsequent purification steps.

Another object of the present invention is to provide highly efficient scalable process for purification of recombinant PTH; comprising a step of diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation and simultaneously increase in optical density followed by subsequent purification steps.

Another object of the present invention is to provide an improved process for purifying recombinant PTH (1-34) by ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation, optionally final diafiltered output can be again concentrated in the continuous manner in same setup to reduce volume handling followed by subsequent purification steps. Another object of the present invention to provide an improved process for purifying recombinant PTH (1-34), wherein said method comprising the steps of:

(a) Solubilization of Inclusion bodies (IBs) of recombinant PTH (1-34);

(b) Refolding;

(c) Ultrafiltration and diafiltration; Wherein ultrafiltration output is diluted with diafiltration buffer before processing through diafiltration;

(d) Cation exchange chromatography - 1;

(e) Enzymatic digestion;

(f) Mixed mode chromatography;

(g) Cation exchange chromatography - II;

(h) Gel filtration chromatography.

Another object of the present invention to provide an improved process for purifying recombinant PTH (1-34), wherein said method comprising the steps of:

(a) Solubilization of Inclusion bodies (IBs) of recombinant PTH (1-34);

(b) Refolding;

(c) Ultrafiltration and diafiltration; Wherein ultrafiltration output is diluted with diafiltration buffer in order to maintain arginine concentration before processing through diafiltration;

(d) Cation exchange chromatography - 1;

(e) Enzymatic digestion;

(f) Mixed mode chromatography;

(g) Cation exchange chromatography - II;

(h) Gel filtration chromatography.

SUMMARY OF THE INVENTION The principal aspect of the present invention is to provide an improved process for purification of protein with decreased protein precipitation or particle formation through ultrafiltration diafiltration (UFDF) followed by subsequent purification steps. Another aspect of the present invention is to provide an improved process for purifying a protein through ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation followed by subsequent purification steps. Another aspect of the present invention is to provide an improved process for purifying recombinant PTH (1-34) by ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation followed by subsequent purification steps. Another aspect of the present invention is to provide highly efficient scalable process for purification of recombinant PTH; comprising a step of diluting output of ultrafiltration three times with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation and simultaneously increase in optical density followed by subsequent purification steps. Another aspect of the present invention is to provide an improved process for purifying recombinant PTH (1-34) by ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation, optionally final diafiltered output can be again concentrated in the continuous manner in same setup to reduce volume handling followed by subsequent purification steps.

Another aspect of the present invention to provide an improved process for purifying recombinant PTH (1-34), wherein said method comprising the steps of:

(a) Solubilization of Inclusion bodies (IBs) of recombinant PTH (1-34);

(b) Refolding;

(c) Ultrafiltration and diafiltration; Wherein ultrafiltration output is diluted with diafiltration buffer before processing through diafiltration;

(d) Cation exchange chromatography - 1;

(e) Enzymatic digestion;

(f) Mixed mode chromatography; (g) Cation exchange chromatography - II;

(h) Gel filtration chromatography.

Another aspect of the present invention to provide an improved process for purifying recombinant PTH (1-34), wherein said method comprising the steps of: (a) Solubilization of Inclusion bodies (IBs) of recombinant PTH (1-34);

(b) Refolding;

(c) Ultrafiltration and diafiltration; Wherein ultrafiltration output is diluted with diafiltration buffer in order to maintain arginine concentration before processing through diafiltration;

(d) Cation exchange chromatography - 1;

(e) Enzymatic digestion;

(f) Mixed mode chromatography;

(g) Cation exchange chromatography - II;

(h) Gel filtration chromatography.

BRIEF DESCRIPTION OF DRAWINGS

Figure-1: represents decrease in turbidity of recombinant PTH (1-34) due to reduction in precipitation / particle formation (aggregate) and simultaneous increase in its optical density (OD).

DETAILED DESCRIPTION OF THE INVENTION

Recombinant human parathyroid hormone PTH (1-34) is a biologically active N-terminal fragment of endogenous human parathyroid hormone (PTH). Therapeutically, PTH (1-34) is used for the treatment of men and postmenopausal women with osteoporosis who are at high risk of fracture. It increases bone mineral density and reduces the risk of vertebral and non-vertebral fractures.

The Parathyroid hormone is a protein of 84 amino acid residues, of which the first 34 amino acids (1-34) are necessary for biological activity. Recombinant human parathyroid hormone (PTH) is a 4117.8 Daltons polypeptide chain consisting of the first 34 amino acids of the parathyroid hormone, with a pi of 8.69. It is produced in the form of a fusion protein by recombinant DNA technology using a genetically engineered E. coli strain. The PTH fusion protein expressed resides in the cytoplasm of E. coli in the form of insoluble inclusion bodies (IBs).

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.

The IBs are released by cell lysis with a high pressure homogenizer and recovered by centrifugation. Several washing and centrifugation steps are performed to wash the IBs off cellular debris, before solubilization. The solubilized IBs are then subjected to refolding following which the fusion partner is separated by proteolysis and PTH ( 1 -34) is purified through a series of chromatographic unit operations.

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.

The downstream process for purifying fusion protein involves solubilization of inclusion bodies, refolding, ultrafiltration diafiltration, digestion and chromatography steps for preparation of drug substance. In the first step of ultrafiltration diafiltration, refolding output is concentrated and buffer exchanged in diafiltration buffer at room temperature. Post ultrafiltration diafiltration, the ion exchange chromatography followed by the digestion of the fusion protein by enzyme is carried out. The subsequent, mixed mode chromatography (MMC) process is used to enable robustness in clearance of product/process related impurities. The successive step of ion exchange chromatography, removes the truncated impurities. Gel filtration chromatography is performed to buffer exchange in the formulation buffer.

The exclusion effect of protein-based pharmaceutical products is dependent upon protein surface charge and concentration; each different protein-based pharmaceutical molecule may require different buffers for diafiltration. The determination of this diafiltration buffer concentration usually requires iterative experiments, necessitating a significant amount of materials and development effort. Thus, reconfiguration with new buffers and amounts of buffers is required every time a new protein purification process is scaled-up for larger scale manufacturing.

The main embodiment of the present invention is to provide an improved process for purification of protein with decreased protein precipitation or particle formation through ultrafiltration diafiltration (UFDF) followed by subsequent purification steps.

Another embodiment of the present invention is to provide an improved process for purifying a protein through ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation followed by subsequent purification steps.

Another embodiment of the present invention is to provide highly efficient scalable process for purification of recombinant PTH; comprising a step of diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation and simultaneously increase in optical density followed by subsequent purification steps.

Another embodiment of the present invention is to provide an improved process for purifying recombinant PTH (1-34) by ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation followed by subsequent purification steps.

Another embodiment of the present invention is to provide an improved process for purifying recombinant PTH (1-34) by ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation, optionally final diafiltered output can be again concentrated in the continuous manner in same setup to reduce volume handling followed by subsequent purification steps.

Another embodiment of the present invention to provide an improved process for purifying recombinant PTH (1-34), wherein said method comprising the steps of:

(a) Solubilization of Inclusion bodies (IBs) of recombinant PTH (1-34);

(b) Refolding;

(c) Ultrafiltration and diafiltration; Wherein ultrafiltration output is diluted with diafiltration buffer before processing through diafiltration;

(d) Cation exchange chromatography - 1;

(e) Enzymatic digestion;

(f) Mixed mode chromatography;

(g) Cation exchange chromatography - II;

(h) Gel filtration chromatography.

Another embodiment of the present invention to provide an improved process for purifying recombinant PTH (1-34), wherein said method comprising the steps of:

(a) Solubilization of Inclusion bodies (IBs) of recombinant PTH (1-34);

(b) Refolding;

(c) Ultrafiltration and diafiltration; Wherein ultrafiltration output is diluted with diafiltration buffer in order to maintain arginine concentration before processing through diafiltration;

(d) Cation exchange chromatography - 1;

(e) Enzymatic digestion;

(f) Mixed mode chromatography;

(g) Cation exchange chromatography - II;

(h) Gel filtration chromatography. The ultrafiltration output obtained as per the present invention is diluted more than one time with diafiltration buffer before processing through diafiltration; preferably three times.

Following are the advantages of the present invention: -

As demonstrated above, the use of proposed strategy of maintaining critical arginine concentration by diluting the concentrated refolding output before passing to the membrane for diafiltration will:

• Help in reducing the shear stress caused to the protein when the arginine is removed at the solid liquid interface or in the entire UFDF operation;

• Decrease protein aggregation/precipitation and particle formation;

• Increase the process recovery;

• Decreases filter size required for filtering the UFDF output thereby reducing the filtration cost;

• Reduce handling volume during operation.

Following flow chart represents method of present invention and traditional method used for UFDF post refolding step.

Present invention

Traditional method

Batch Traditional method Present invention

Scale 30 L refolding 30 L refolding

Turbidity (NTU)

Sample

BF AF BF AF

SIB 7.39 3.74 4.49 2.20

RF OP 2.05 1.82 1.08 0.94

UF OP (Before 8.98 15.2

dilution)

UF OP (After NA 66

dilution)

DV (1) 158 188

DV (2) 542 117

DV (3) 465 73.3

DV (4) 376 47.6

DV (5) 288 38.6

UF DF output 283 38.6

Table 1: Comparison between method of present invention and traditional method used for

UFDF post refolding step

As it can be seen from table 1, method of present invention results in reduction of protein precipitation or particle formation by ~7 folds. Due to use of present invention during UFDF, the output of subsequent chromatography step increases by 20-30% thereby increasing the yield.

The present invention is an efficient and scalable process for purification of recombinant PTH (1-34) as by following present invention there is increase of more than 20% productivity after MMC output.

The invention will now be further described by the following example, which is illustrative rather than limiting. The following example illustrate the purification of rPTH (1-34) described in the present invention and the means of carrying out the invention in order to obtain decrease precipitation or particle formation and simultaneously increase in optical density of rPTH (1-34). EXAMPLES

Example 1

Recombinant PTH (1-34) is used as a model protein to demonstrate present invention. rPTH (1- 34) was produced in the form of a fusion protein by recombinant DNA technology using a genetically engineered E. coli strain. The rPTH (1-34) fusion protein expressed resides in the cytoplasm of E. coli in the form of insoluble inclusion bodies (IBs). The IBs were released by cell lysis with a high pressure homogenizer and recovered by centrifugation. Several washing and centrifugation steps were performed to wash the IBs off cellular debris, before solubilization. The solubilized IBs were then subjected to refolding. After refolding ultrafiltration was carried out and ultrafiltration output was diluted three times with diafiltration buffer before processing through diafiltration to maintain arginine concentration. Optionally, final diafiltered output can be again concentrated in the continuous manner in same setup to reduce volume handling. Ultrafiltration Diafiltration output was further subjected to cation exchange chromatography - I (CEX-I) followed by enzymatic digestion through use of Enterokinase, mixed mode chromatography (MMC), cation exchange chromatography - II (CEX-II) and gel filtration chromatography (GFC). Use of present invention resulted in reduction of protein precipitation or particle formation by ~7 folds (Table 1 and Figure 1 ) and due to use of present invention during UFDF, the output of subsequent chromatography step increased by 20-30% thereby increasing the yield compared to traditional method (Table 2 and Table 3).

Traditional method

Batch 1 Batch 2 Batch 3

Sample Average TOD

Total OD (TOD) at 280 nm

at 280 nm

Amount of starting IB

90 90 90 90

(g)

Solubilized IB 24912 23400 23580 23964

Refolding Output 32436 31164 31927 31842

UFDF Output 36147 32229 36704 35027

CEX-I input 8255 8748 7945 8316

CEX-I Output 4842 4761 4780 4794

MMC Input 4828 4734 4771 4778

MMC Output 745 746 727 739

Table 2: Average total optical densities obtained by traditional method

Table 3: Average total optical densities obtained by present invention The increase in productivity at CEX-I output and at MMC output was respectively 31.31% and 23.08% represents the effectiveness of highly scalable present purification process of rPTH (1- 34) in comparison with traditional method. The comparison of increase in productivity was done at CEX-I output and MMC output step as CEX-I was immediate step after UFDF 1 and purified the fusion protein. MMC purified the cleaved PTH from the fusion protein. There was an enzymatic digestion step between CEX-I and MMC in which PTH was cleaved from fusion tag through use of Enterokinase enzyme.

The TOD value of UFDF Output was higher in traditional method than in present invention because the traditional batches had higher turbidity.