DESC:RELATED APPLICATION

This application is related to Indian Provisional Application 202121004831 filed on 4th Feb, 2021 and is incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to an improved process for the purification of proteins. More particularly, the present invention pertains to a process for purification of Pegfilgrastim (PEG-GCSF). The present invention further pertains to a purification process using dual UFDF / using ultrafiltration diafiltration step prior to CEX chromatography step.

BACKGROUND OF THE INVENTION

GCSF is a human endogenous secretory protein which selectively induces the development of granulocyte committed progenitors from multipotent hematopoietic cells. The term "Peg-GCSF" or "pegylated GCSF" refers to a GCSF protein which is covalently linked with one or more polyethylene glycol moieties.

Pegfilgrastim is a covalent conjugate of recombinant methionyl human G-CSF (filgrastim) and monomethoxypolyethylene glycol. Filgrastim is a water-soluble 175 amino acid protein with a molecular weight of approximately 19 kilodaltons (kD). Filgrastim is obtained from the bacterial fermentation of a strain of E coli transformed with a genetically engineered plasmid containing the human G-CSF gene. To produce pegfilgrastim, a 20 kD monomethoxypolyethylene glycol molecule is covalently bound to the N-terminal methionyl residue of filgrastim. The average molecular weight of pegfilgrastim is approximately 39 kD.

To ensure the safety of biopharmaceuticals, regulatory agencies impose stringent purification standards and relevant quality attributes (e.g. identity, purity and biological activity) for recombinant proteins intended for human administration. Common standards require that protein-based therapeutic products are substantially free from impurities, including product related contaminants, such as aggregates, fragments, and inactive variants of the target recombinant protein, and process related contaminants, such as leached chromatography resin ligands, culture media components, host cell proteins and nucleic acids, viral contaminants, and endotoxins.

Efficient and economic large scale purification of proteins, e.g., therapeutic proteins including antibodies, is an increasingly important consideration for the biotechnology and pharmaceutical industries. As proteins are typically produced using cell culture methods, e.g., using either mammalian or bacterial host cells engineered to produce proteins of interest in appropriate media, processes for purifying proteins include many different steps to remove impurities from the media components and host cells. Although processes for purifying proteins may vary depending on the properties of a particular protein of interest under purification, the processes typically include at least steps of recovering the protein from host cells and/or cell debris, e.g., using centrifugation and/or filtration methods, and steps for purifying the proteins, e.g., using one or more chromatography and/or filtration methods, to separate the protein from various impurities.

In last few years development and manufacturing of therapeutic G-CSF involves numerous purification schemes. European patent EP2341061, discloses a purification process involving a series of four chromatography steps, comprising two gel filtration chromatography, a cation exchange chromatography and an anion exchange chromatography for preparation of G-CSF.

Another European patent EP1904522, discloses a purification process used for purification of G-CSF primarily involving three chromatography steps namely, two cation exchange chromatography and a hydrophobic interaction chromatography.

US10519209 discloses a purification process used for purification of PEG GCSF by cation exchange chromatography followed by ultrafiltration.

In light of the above, new methods are needed which are effective in purifying PEG-GCSF with reduced precipitation or particle formation. Thus, there is still an unmet need for improved production methods that are cost-effective, stable for high recovery of PEG-GCSF and amenable for large-scale production.

OBJECTS OF THE INVENTION

The principal object of the present invention is to provide an improved process for separation of residual/unreacted moieties like mPEG-PAL 20, sodium cyanoborohydride and filgrastim from the pegylated filgrastim molecules using UFDF 1 prior to CEX chromatography step.

Another object of the present invention is to provide an improved process for separation of residual/unreacted moieties like mPEG-PAL 20, sodium cyanoborohydride and filgrastim from the pegylated filgrastim molecules using dual UFDF step.

Another object of the present invention is to provide an improved process for purification of PEG-GCSF using UFDF 1 prior to CEX chromatography step to lower viscosity of load solution for CEX chromatography step through which back pressure in CEX chromatography column can be controlled.

Another object of the present invention is to provide an improved process for purification of PEG-GCSF using UFDF 1 prior to CEX chromatography step to avoid the air bubble entrapment in the CEX chromatography column which could cause the disturbance in the packed bed of column ultimately impacting the resolution of different product related impurity.

SUMMARY OF THE INVENTION

The principal aspect of the present invention is to provide an improved process for separation of residual/unreacted moieties like mPEG-PAL 20, sodium cyanoborohydride and filgrastim from the pegylated filgrastim molecules using UFDF 1 prior to CEX chromatography step.

Another aspect of the present invention is to provide an improved process for separation of residual/unreacted moieties like mPEG-PAL 20, sodium cyanoborohydride and filgrastim from the pegylated filgrastim molecules using dual UFDF step.

Another aspect of the present invention is to provide an improved process for purification of PEG-GCSF using UFDF 1 prior to CEX chromatography step to lower viscosity of load solution for CEX chromatography step through which back pressure in CEX chromatography column can be controlled.

Another aspect of the present invention is to provide an improved process for purification of PEG-GCSF using UFDF 1 prior to CEX chromatography step to avoid the air bubble entrapment in the CEX chromatography column which could cause the disturbance in the packed bed of column ultimately impacting the resolution of different product related impurity.

BRIEF DESCRIPTION OF DRAWINGS

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with its detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:

Figure 1: Process Flow Diagram of Manufacturing Process For PEG-GCSF
Figure 2: Purity Results of Peg Mix OP and UFDF 1 OP
Figure 3: Comparison of Input Vs Output of Process Related Impurity
Figure 4: Percentage Clearance of Process Components in UFDF 1 Step
Figure 5: CEX Column Back Pressure Trend during Loading of CEX Input

DETAILED DESCRIPTION OF THE INVENTION

There is always need to have robust, consistent and an improved process for the production of PEG-GCSF at commercial scale for the human use which can cater the market demand to the unmet need of patients.

The principal embodiment of the present invention is to provide an improved process for separation of residual/unreacted moieties like mPEG-PAL 20, sodium cyanoborohydride and filgrastim from the pegylated Filgrastim molecules using ultrafiltration diafiltration 1 (UFDF 1) prior to cation exchange chromatography step.

Another embodiment of the present invention is to provide an improved process for separation of residual/unreacted moieties like mPEG-PAL 20, sodium cyanoborohydride and filgrastim from the pegylated filgrastim molecules using dual UFDF step.

Another embodiment of the present invention is to provide an improved process for purification of PEG-GCSF using UFDF 1 prior to CEX chromatography step to lower viscosity of load solution for CEX chromatography step through which back pressure in CEX chromatography column can be controlled.

Another embodiment of the present invention is to provide an improved process for purification of PEG-GCSF using UFDF 1 prior to CEX chromatography step to avoid the air bubble entrapment in the CEX chromatography column which could cause the disturbance in the packed bed of column ultimately impacting the resolution of different product related impurity.

Another embodiment of the present invention is to provide an improved process for purification of Pegylated Filgrastim comprising:
a. Pegylation Step;
b. Ultrafiltration Diafiltration – 1;
c. Cation Exchange Chromatography;
d. Ultrafiltration Diafiltration – 2; and
e. 0.22µM Filtration.

Detailed process flow diagram of improved process is given in Figure 1.

During pegylation reaction, mPEG is covalently linked with filgrastim molecules (site specific pegylation) in presence of sodium cyanoborohydride. Presence of unreacted filgrastim, mPEG PAL and sodium cyanoborohydride will create the high back pressure and disturbance in the column bed due to the chemical nature of the reagents. There is high probability of failure of batches at higher scale due to viscous load solution (in presence of unreacted mPEG, filgrastim and sodium cyanoborohydride).

Hence to remove the unwanted moieties before loading onto the CEX chromatography column, ultrafiltration and diafiltration step (with 30 kDa ultrafiltration membrane) is added after the pegylation step. Diafiltration with acetate buffer will further reduce the concentration of unreacted mPEG (20 kDa), unreacted filgrastim (19.2 kDa) and dissolve sodium cyanoborohydride. Eventually reduce the burden on the CEX chromatography column and will improve the performance of CEX chromatography column.

% Unreacted Filgrastim by SE-HPLC analysis
Process Stage
PEG GCSF purification process not having UFDF 1 prior to CEX chromatography step
PEG GCSF purification process having UFDF 1 prior to CEX chromatography step

Batch no. P5.3-BM-0154 P5.3-BM-0155 P5.3-BM-0156 P5.5-BM-0001 P5.5-BM-0002 P5.5-BM-0003
% Native filgrastim in Pegylation Output 9.08 7.38 8.00
6.53 10.31
10.17

% Native filgrastim in UFDF 1 Output NA NA NA 1.22 1.26
1.25

% Reduction of native Filgrastim from pegylation to UFDF 1 step NA NA NA 81.32
87.78
87.71

Table 1: Comparison between purification process not having UFDF 1 prior to CEX step and having UFDF 1 prior to CEX step for reduction of unreacted filgrastim from pegylated filgrastim.

As it can be seen from table 1, process of present invention (i.e using UFDF 1 prior to CEX step) results in drastic reduction (>80%) of unreacted filgrastim from pegylated filgrastim.

Back pressure (delta P) of column during loading of SP input onto CEX column
Process Stage PEG GCSF purification process not having UFDF 1 prior to CEX chromatography step. PEG GCSF purification process having UFDF 1 prior to CEX chromatography step.
Batch No. P5.3-BM-0154 P5.3-BM-0155 P5.3-BM-0156 P5.3-BM-0001 P5.3-BM-0002 P5.3-BM-0003
Min column back pressure during loading phase (Delta P) 0.1 0.1 0.1 0.06 0.04 0.08
Max column back pressure during loading phase (Delta P) 1.60 1.70 1.40 1.17 1.02 1.10
Average column back pressure during loading phase (Delta P) 1.50 1.50 1.20 0.89 0.92 1.00

Table 2: Comparison between purification process not having UFDF 1 prior to CEX step and having UFDF 1 prior to CEX step for average column back pressure during loading phase (delta P) onto CEX column.

As it can be easily understood from table 2, process having UFDF 1 prior to CEX step results in reduction of back pressure onto CEX column. As a result of this, risk of failure of batch due to high back pressure could be avoided. This will also reduce the frequency of repacking of CEX chromatography due to back pressure.

The term “PEGylation” refers to the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG) polymer chains to molecules and macrostructures, such as a drug or therapeutic protein, which is then described as PEGylated. PEGylation improves drug solubility and decreases immunogenicity. PEGylation also increases drug stability and the retention time of the conjugates in blood, and reduces proteolysis and renal excretion, thereby allowing a reduced dosing frequency.

The term “ultrafiltration” refers to membrane filtration (Tangential Flow filtration) technique which employs controlled pore size, semi permeable membranes to concentrate or fractionate dissolved molecules. Molecules much larger than the pores are retained in the feed solution and are concentrated in direct proportion to the volume of liquid that passes through the membrane. Molecules having a size which is close to the pore size of the membrane concentrate to a lesser extent with some of the molecules passing through the membrane in the permeate. The concentration of freely permeable molecules (salts) in the sample remains essentially unchanged. Membranes suitable for ultrafiltration (referred to as ultrafiltration or UF membranes) are defined by the molecular weight cut-off (MWCO) of the membrane used. Ultrafiltration can be applied in cross-flow or dead-end mode.

The term “diafiltration” refers to a technique that uses ultrafiltration membranes to completely remove, replace or lower the concentration of salts or solvents from solutions containing proteins, peptides, nucleic acids, and other biomolecules. The process selectively utilizes permeable (porous) membrane filters to separate the components of solutions and suspensions based on their molecular size. An ultrafiltration membrane retains molecules that are larger than the pores of the membrane while smaller molecules such as salts, solvents and water, which are 100% permeable, freely pass through the membrane. During diafiltration, buffer is introduced into the recycle tank while filtrate is removed from the unit operation. In processes where the product is in the retentate, diafiltration washes components out of the product pool into the filtrate, thereby exchanging buffers and reducing the concentration of undesirable species. When the product is in the filtrate, diafiltration washes it through the membrane into a collection vessel.

Advantages of present invention:

As disclosed above, the use of proposed an improved process of using UFDF 1 just prior to CEX chromatography step will:

• Help in removal of unwanted moieties before loading onto the CEX chromatography column
• Reduce the burden on the CEX chromatography column (as a result of which the risk of failure of batch due to high back pressure can be neglected) and
• Avoid the air bubble entrapment in the CEX chromatography column.

The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also form the part of this specification and are intended to be included within the scope of it.

EXAMPLES

EXAMPLE 1: PEGFILGRASTIM PURIFICATION WITH DUAL UFDF STEP

Pegylation:

Purified Filgrastim was covalently linked with the mono methoxy polyethylene glycol 20 kDa (mPEG) to form Pegfilgrastim molecules. Aldehyde group of mPEG molecule reacted with NH2 group of N terminal methionine to create Schiff base which was converted to secondary amine upon reduction by sodium cyanoborohydride. With the site specific Pegylation, there was possibility of nonspecific attachment of mPEG molecule with NH2 group of lysine (amino acid) which generated dipegylated and multipegylated species with the monopegylated species (Target moiety). For Pegylation reaction parameters like pH of Pegylation mixture (pH 5.0), concentration of Filgrastim (1 mg/mL) were important factors for Pegylation efficiency. Pegylation efficiency was monitored by analytical technique like purity by SE-HPLC for Pegylation mixture (Pegylation Output).

Ultrafiltration and Diafiltration - 1:

Pegylated mixture was concentrated and diafiltered with buffer to bring the condition like pH and conductivity required for the next step (Chromatography step) of purification process using the flat plate module of TFF (Cassettes with MWCO -30 kDa). This step removed the unreacted chemicals like Filgrastim, mPEG and sodium cyanoborohydride through size based separation. Pegylated species of Filgrastim retained on the retentate side of membrane while unreacted chemical and protein pass through membrane to permeated size based on size of molecules. For Ultrafiltration and diafiltration parameters like concentration factor (4-5X), TMP (10 psi) were important factors for process operation. Process performance of ultrafiltration and diafiltration 1 step monitored through measuring purity SE-HPLC and optical density.

Pegylation output and UFDF-1 output samples of four batches (P5.5-MB-0020, P5.5-MB-0022, P5.5-MB-0023 and P5.5-MB-0024) were evaluated for clearance of impurities in UFDF 1 output. Detailed result of evaluation is given in Table 3.

Process Step Sample Name Test P5.5-BM-0020 P5.5-BM-0022 P5.5-BM-0023 P5.5-BM-0024
Result (%) Result (%) Result (%) Result (%)
Pegylation Output Peg Mix OP SE-HPLC (%) 85.00 84.00 85.00 81.00
Ultrafiltration and Diafiltration 1 OP UFDF 1 OP Total Cyanide (ppm) 390.8 0.9 3.8 251.5
Unreacted mPEG PAL (µg) 133.2 157.1 132.8 136.2
SE-HPLC (%) 85.80 85.25 83.47 85.40

Table 3: Pegylated Mixture Output and UFDF-1 output results Represents Similarity in Terms of Purity of Product

Based on the above SE-HPLC data, Pegylation output and UFDF 1 output purity by SE-HPLC shows consistent purity in four batches. (Figure 2)

Further to show the clearance of free Filgrastim, unreacted mPEG PAL and residual cyanoborohydride in UFDF-1 step output data was evaluated.

Molecule Name Batch Number Quantity Observed in Pegylation OP Quantity Observed in UFDF 1 OP % Reduction

% Free Filgrastim in POP P5.5-BM-0020 6.75 0.45 93.33
P5.5-BM-0022 7.70 0.59 92.34
P5.5-BM-0023 3.32 0.24 92.77
P5.5-BM-0024 11.71 0.68 94.19
Total Cyanide (ppm) P5.5-BM-0020 1256.8* 390.8 68.91
P5.5-BM-0022 1256.8* 0.9 99.93
P5.5-BM-0023 1256.8* 3.8 99.70
P5.5-BM-0024 1256.8* 251.5 79.99
Total mPEG PAL (g) P5.5-BM-0020 3062.23 0.0001332 99.99999565
P5.5-BM-0022 3107.91 0.0001571 99.99999495
P5.5-BM-0023 3079.33 0.0001328 99.99999569
P5.5-BM-0024 2649.79 0.0001362 99.9999486
Table 4: Assessment Results of Free Filgrastim, Unreacted mPEG PAL and Residual Cyanoborohydride after UFDF-1 step

From above data it shows significant amount of process related components are being cleared during UFDF 1 step. (Figure 3)

The UFDF 1 OP shows above 90% clearance of free Filgrastim in all the batches.
The UFDF 1 OP shows 100% clearance of unreacted mPEG PAL in all the batches.

The UFDF 1 OP shows nearly 100% clearance of total cynide in P5.5-BM-0022 and P5.5-BM-0023, where in the clearance in P5.5-BM-0020 and P5.5-BM-0024 is ~69% and ~80%. The overall percentage reduction of free Filgrastim, unreacted mPEG PAL and residual cyanoborohydride in UFDF 1 step is shown in Figure 4.

Additionally column back pressure data was also evaluated for batches where UFDF 1 is not part of the routine process and where UFDF 1 step is part or executed in process. Refer Table 5 which shows column back pressure trend during loading onto a column.
Parameter Batch no for Small Scale Process not having UFDF 1 step Batch no for Small Scale Process having UFDF 1 step Batch no for Large Scale Process having UFDF 1 step
P5.3-BM-0154 P5.3-BM-0155 P5.3-BM-0156 P5.3-BM-0157 P5.3-BM-0158 P5.5-BM-0001 P5.5-BM-0002 P5.5-BM-0003
Column Back Pressure During Protein Loading Phase (Delta P in bar) Minimum 0.1 0.1 0.1 0.1 0.1 0.06 0.04 0.08
Maximum 1.60 1.70 1.40 1.20 1.20 1.17 1.02 1.10
Average 1.50 1.50 1.20 1.10 1.10 0.89 0.92 1.00

Table 5: Back Pressure (Delta P) of column during loading of CEX input onto CEX Column

Column back pressure trend during loading of CEX input in Table 5 is respresented by Figure 5.

The consistent reduction in column back pressure value post UFDF 1 step in small and large scale operation illustrated the clearance of (free Filgrastim, unreacted mPEG PAL and residual cyanoborohydride) from the UFDF 1 step output.

Ion Exchange Chromatography:

Nonspecific Pegylation of Filgrastim molecules generated product related impurities for Pegfilgrastim molecules. Cation exchanger resin purified the Pegfilgrastim molecule (monomer) by separating product related impurities by selective elution of impurity and target proteins. For ion exchange chromatography step parameters like pH of input (4.00), load factor 19 mg/ml of resin and elution conductivity (12 mS/cm) were important factors for process operation. Process performance of chromatography step monitored through measuring purity SE-HPLC, RP-HPLC, CEX-HPLC and optical density.

Ultrafiltration and Diafiltration - 2:

Purified Pegfilgrastim was concentrated and diafiltered with buffer to bring the condition like pH and conductivity required for the next step (0.22 µM filtration) of purification process using the flat plate module of TFF (Cassettes with MWCO -30 kDa). This step removed the unreacted chemicals like sodium chloride through size based separation and brought the conditions required for the formulation. Pegfilgrastim molecule retained on the retentate side of membrane. While unreacted chemical pass through the membrane to permeated. For Ultrafiltration and diafiltration parameters like concentration factor (4-5X), TMP (10 psi) were important factors for process operation. Process performance of ultrafiltration and diafiltration step monitored through measuring purity SE-HPLC, RP-HPLC and optical density

0.22 µM Filtration:

To control microbiological factors like bioburden, purified Pegfilgrastim molecules having the condition required for formulation was filtered through 0.22 µM filter for the reduction of microbial count in the process intermediates. For 0.2µM filtration step parameters like Flow rate (1 L/min), TMP (10 psi) were important factors for process operation. Process performance of ultrafiltration and diafiltration step monitored through testing filter integrity. ,CLAIMS:We Claim,

1. A process for separation of residual/unreacted moieties from the Pegylated fFilgrastim molecules comprising ultrafiltration diafiltration 1 (UFDF 1) prior to cation exchange chromatography step.

2. The process according to claim 1, wherein residual/unreacted moieties are mPEG-PAL 20, sodium cyanoborohydride and Filgrastim.

3. The process according to claim 1, wherein UFDF 1 prior to cation chromatography step to lowers the viscosity of load solution for cation chromatography step through which back pressure in cation chromatography column can be controlled.

4. The process according to claim 1, wherein UFDF 1 prior to cation chromatography step avoids the air bubble entrapment in the cation chromatography column.

5. A process for separation of residual/unreacted moieties from the Pegylated Filgrastim molecules comprising dual ultrafiltration diafiltration step.

6. A process for purification of Pegylated Filgrastim comprising:
a. Pegylation Step;
b. Ultrafiltration Diafiltration – 1;
c. Cation Exchange Chromatography;
d. Ultrafiltration Diafiltration – 2; and
e. 0.22µM Filtration.