FIELD OF THE INVENTION
The present invention provides a stable formulation for recombinant human Granulocyte - Colony Stimulating Factor (G-CSF) in the pH range of 4.0 to 5.0 and a conductivity value greater than 1.0 milliSiemens/cm, comprising of L-methionine, to suppress the formation of oxidized G-CSF during storage. Good storage stabilities at higher conductivities and pH were achievable without the addition of stabilizer proteins of human or animal origin. This invention also includes the process for preparing stable G-CSF formulation
BACKGROUND OF THE INVENTION
Granulocyte - colony stimulating factor (G-CSF) is a hematopoietic growth factor that stimulates the proliferation and differentiation of hematopoietic precursor cells and activation of mature neutrophils. G-CSF is capable of supporting neutrophil proliferation in vitro and in vivo. Large quantities of recombinant G-CSF have been produced in genetically engineered Escherichia coli and have been successfully used in the clinic to treat cancer patients suffering from chemotherapy-induced neutropenia. E. coli produced G-CSF is a 175 amino acid polypeptide chain containing an extra methionine at its N-terminus. This protein has been produced by expressing a G-CSF gene in E. coli and purifying the protein product to homogeneity. It is a hydrophobic protein that has five cysteine residues, four of which are involved in disulphide bonding. The free cysteine residue is generally implicated in the formation of higher molecular weight aggregates upon storage in solution. Aggregates of the proteins can also be formed from oxidized forms of the protein that arise by oxidation of the internal methionine residues in the primary sequence of the protein. Out of the four methionine residues, one is at the N-terminus and the other three are internal. The oxidized forms of the protein that contain oxidized methionine at position 122 can be separated from the forms containing oxidized methionines at positions 127 or 138 and the native protein by regular reverse phase HPLC separation procedures.
Several pharmaceutical preparations of G-CSF have been described in earlier patents that are suitable for administration to humans. The emphasis in various formulation strategies is on preventing the formation of higher order aggregates of the protein by disulphide exchange involving the free cysteine residue. Besides, the free cysteine residue, the occurrence of methionine residues that can undergo oxidation in the presence of free radicals in solution, also contribute to the formation of altered conformational forms of G-CSF that can initiate the formation of higher order aggregates of the protein in solution. A more effective formulation for G-CSF would be the one that includes L-methionine to suppress the oxidation of internal methionine residues (at positions 122, 127, 138) in the G-CSF sequence formulated in a buffer of suitable pH and conductivity value that maintains the structural integrity of the molecule.
One of the earlier formulation patents for G-CSF by Boone, Thomas (Amgen) in EP 0373679, has described the use of a pharmaceutically effective amount of the protein in

an acidic solution or buffer at low pH and conductivity values. The pH value described is in the range of 2.75 to 4.0 and the conductivity value is less than 1000 m mhos/cm. The formulation excipients comprise of a tonicity modifier like 5% mannitol and a non-ionic surfactant, and the authors describe this formulation as one where minimal aggregation occurs for the protein.
In US 5919757 (granted in 1999), Uwe Michaelis, (Boehringer Mannheim) describes an aqueous pharmaceutical preparation of G-CSF comprising of a therapeutic amount of the protein in a buffer selected from the group of citrate, maleate, phosphate and arginine or their salts thereof and a surfactant at a pH value between 7.0 and 8.0. This formulation is described as one with a long shelf life, which can tolerate mechanical stress and has a physiologically compatible pH value. In a patent by Chugai Seiyaku Kabushiki Kaisha GB 2193631, a stable formulation of G-CSF is described that contains besides the active ingredient, at least one substance selected from a pharmaceutically acceptable surfactant, saccharide, protein and a high molecular weight compound.This formulation is described as being highly stable and capable of preventing the loss of active component from solution by adsorption to container surfaces, association, polymerization or oxidation of the said component.
In US 5919443 assigned to Boehringer Mannheim formulations of lyophilized pharmaceutical preparations of G-CSF are described, that contain various sugars as stabilizing agents. In addition, the invention is concerned with a process for the production of stabilized lyophilisates, as well as the use of maltose, raffinose, sucrose, trehalose or amino sugars as stabilizing agents of pharmaceutical agents containing G-CSF.
The patent assigned to Rhone-Poulenc Rorer S.A. in US 5554150, methods and devices are described for the delivery of G-CSF to the patients. The method comprises continuous sub-cutaneous delivery of a solution comprising granulocyte colony stimulating factor to the patient. Devices for continuous administration are disclosed as well as the preferred composition of the solution which is also described in patent GB 2163631. The solution consists of G-CSF, serum albumin, non-ionic surface active agent, saccharide, disodium phosphate, monosodium phosphate, sodium chloride and water.
In US 5104651 and EP 0373679, Boone; Thomas C and Kenney; William C have described methods to obtain stable pharmaceutically acceptable formulations of hydrophobic proteins. The said formulation has an effective amount of hydrophobic protein like G-CSF or IL-2 at acidic pH and advantageously a low conductivity which is less than lOOO.mu.mhos/cm. Advantageously, the pH is about 2.5 to 4.0 and in a preferred embodiment, no buffer is present. The method involves combining the protein advantageously with acid without the addition of a salt to make a pharmaceutically acceptable formulation. Optionally, it can also contain a tonicity modifier.
In a European patent filed by Chugai Seiyaku Kabushiki, EP 1197221 describes a stable lyophilized, CHO-cell derived G-CSF formulation in the pH range of 5.0 - 7.0, more specifically around 6.5 and containing mannitol, Tween 20 and amino acids. The amino

acids are one or more selected from a group of lysine, histidine, arginine, aspartic acid and glutamic acid; and one or more amino acids selected from a group consisting of phenylalanine, tryptophan and leucine; and with methionine. The formulation is claimed to have a residual ratio of G-CSF of 90% or more after long term storage testing and methionine - oxidized G-CSF of 1% or less.
SUMMARY OF THE INVENTION
The present invention provides a stable aqueous formulation comprising
a pharmaceuticallyeffective amount of therapeutic recombinant human
Granulocyte -Colony Stimulating Factor (G-CSF) protein in the range of
0.2 to 0.8 mg/ml
a tonicity modifier like mannitol or sorbitol in the range of 20 to 70
mg/ml,
a surfactant like Polysorbate20 or Polysorbate80 in the concentration
range of 0.001 to 0.01% and
an amino acid, which is an oxidation suppressant for the methionine
residues in the protein in the concentration range of 0.05 to 0.5mg/ml
in an acidic buffer in the conductivity range greater than one milliSiemens per cm
and a pH range of 4.0 to 5.0.
The present invention also provides a process for preparing a stable G-CSF formulation comprising the steps of formulating the:
purified protein G-CSF in the concentration range of 0.2 to 0.8 mg/ml; adding a tonicity modifier in die range of 20 to 70 mg/ml; adding a surfactant in the concentration range of 0.001 to 0.01%; in an acidic buffer in the conductivity range greater than one milliSiemens per cm and a pH value in the range of 4.0 to 5.0.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a RP-HPLC separation of recombinant human G-CSF from its oxidized forms. The form of G-CSF with methionine 122 oxidised has a retention time around 26 minutes, whereas the form having methionines at 127 and 138 have a retention time of around 32 minutes on a C-18 RP-HPLC column. The intact G-CSF molecule, with no methionines oxidized, has a retention time of about 33 min on the same column. The solvent system used is acetonitrile /TFA. A) With methionine addition B) Without methionine addition
DETAILED DESCRIPTION OF THE INVENTION
A method is described for formulating an aqueous composition of Granulocyte - Colony Stimulating Factor (G-CSF). Herein, the term G-CSF refers to a protein that has the sequence of the naturally occurring human G-CSF or the recombinant form with or without an additional N-terminal methionine residue. It can also encompass derivatives of

the molecule which are structural analogues or derivatives with amino acid additions or deletions to the naturally occurring G-CSF molecule.
According to the present invention, G-CSF is formulated in a buffer of acidic pH comprising of a tonicity modifier, a surfactant and an amino acid, which is an oxidation suppressant for the methionine residues in the protein. The buffers chosen are in the conductivity range of 1.0 to 6.0 milliSiemens, more preferably in the 1.1 to 2.0 milliSiemens range and a pH range of 4.0 to 5.0. Various buffer substances can be used in the formulation like acetic acid, phosphoric acid, citric acid or lactic acid and they can be present either in the free acid form or as salts of alkali, alkaline earth metals or ammonia. The more preferred substances being acetate and phosphate. The desired pH value is obtained by the addition of base or acid as required for attaining the final pH value. In the pH range of 3.0 to 6.0, more preferably in the range of 3.5 to 5.5, a preferred embodiment being the range of pH 4.0 to 5.0. The conductivities chosen are such that the values are suitable for therapeutic administration. The concentrations of buffer solutions that can be used are in the range of 1 to 200mM, but more preferably in the 15 to 150mM range of concentration and most preferably in the 40 to 100 mM range. Besides the buffer substance the formulation also contains other excipient substances that provide an isotonicity that is close to the physiological levels in the human body. The necessary isotonicity value is conferred by the use of neutral auxiliary substances like sugars and sugar alcohols. These are non-ionic and extremely well-soluble in water at high concentrations. Examples of such substances are mannitol, sorbitol, glycerol and other similar sugars and sugar alcohols.
In a preferred embodiment of the invention, an oxidation retarding agent is also added to the formulation buffer to prevent the formation of multiple oxidized forms of G-CSF. The amino acid sequence of the G-CSF molecule is known to have three methionine residues, at positions 122, 127 and 138. The oxidized forms of G-CSF are formed by the oxidation of one or more of these methionine residues in the protein. Reverse phase HPLC methods are used to separate and identify the various methionine oxidized forms of G-CSF which arise during purification, formulation or storage. The oxidation state of a protein after formulation remains stable, if the protein is not exposed to an oxidizing environment during long term storage. However, in the case of G-CSF and other protein formulations containing oxidants, protein stability can be maintained throughout the shelf life of the product, by the addition of specific amino acids capable of protecting the protein from the damaging effects of oxidative free radicals. For example, certain lots of G-CSF have been shown to exhibit an increased tendency to produce oxidized forms. This was predominantly due to a non-ionic surfactant Polysorbate (present in the formulation buffer) having a tendency to produce peroxides, that cause oxidation of methionine residues in the protein. Polysorbate, a common excipient in the therapeutic formulations of G-CSF, is generally added in liquid formulations to eliminate the formation of particulates that are occasionally observed at elevated temperatures. It has been reported by earlier workers that the peroxide content of Polysorbate had a greater effect on protein oxidation than atmospheric oxidation in the vial headspace or the effects of product foaming in the fill lines The reports also mention a definite correlation between the peroxide levels in Polysorbate and the degree of oxidation, to the extent that

the oxidation of methionines was accelerated (<1 day) by formulating G-CSF at very high Polysorbate concentrations (0.05 - 0.10%). As expected, the extent of oxidation varied depending upon the contaminating levels of peroxides present in a given lot of Polysorbate (pp303-328 of Formulation, Characterization, and Stability of Protein Drugs - Case Histories, Edited by Rodney Pearlman and Y. John Wang, 1996, Plenum Press, New York).
Addition of the amino acid methionine to the protein in the formulation buffer helps in preventing the attack of the oxygen free radicals, produced chiefly, but not limited to, the peroxides contaminating commercial Polysorbate preparations. Methionine can be added as a free amino acid or as its salt containing the D, L' or DL forms. The L- variant is however, preferred. The preferred final concentration is in the range of 0.01 to 1.0 mg/ml and more preferably in the 0.05 to 0.5 mg/ml. Formulations of the present invention may contain in addition to methionine, other agents like non-ionic surfactants and sugars. The non-ionic surfactant preferred in this case is from the sorbitan fatty acid ester and polyoxyethylene sorbitan fatty acid esters series, that are added in the concentration range of 0.001 to 0.01%. The sugar molecule preferred, belongs to the group of sugar alcohols consisting of molecules such as sorbitol and mannitol. The concentration range of the sugars is in the 5-100 mg/ml, the more preferred range being 20 to 70 mg/ml.
The active ingredient in this invention can be cytokines, more preferably Granulocyte-Colony Stimulating Factor, in its natural or recombinant form, derivatives of G-CSF or functional polypeptides of the same molecule. The concentration of the active substance is in the range of 0.1 to 1.0 mg/ml more preferably in the range of 0.2 to 0.8 mg/ml.
EXAMPLE 1
Methodology for preparation of formulated G-CSF: Purified G-CSF protein or its derivative is buffer exchanged with 40mM to lOOmM concentration of the formulation buffer in the pH range of 4.0 to 5.0. To this, excipients are added in stated amounts either in solid form or from higher molarity stock solutions. Sugars or sugar alcohols like sorbitol or mannitol are preferably added in solid form at a concentration of 20 - 70 mg/ml. Surfactants are added at a concentration of 0.001 to 0.01 % either directly or from a freshly prepared stock solution in formulation buffer of the surfactant at a higher molarity. The amino acid methionine is also added from a stock solution to give a final concentration in the range of 0.05 to 0.5 mg / ml. The excipient substances chosen are of the formulation grade and essentially free of endotoxins. The formulated protein solution is stirred gently at room temperature to ensure a thorough and homogenous mixture of the contents. The final pH and conductivity values are checked and re-adjusted to the desired value, if required, by the addition of small amounts of acid or base component of the buffer. The solution so obtained is filtered through a 0.22 micron or similar sterilizing grade filtration membrane and filled into suitable containers like vials or syringes for ease in therapeutic administration.

EXAMPLE 2
Methodology for stability testing of formulated G-CSF: This example relates to the stability indicating tests for different formulations of G-CSF which are held at defined storage temperatures. The temperature parameters are set, such that the accelerated and real time stability of the formulated protein can be ascertained.
The stability determining parameters are initially defined and various biochemical and biological tests are conducted at different intervals of time to ensure that the quality of the protein falls within the defined specifications. A very useful test method to determine the presence of aggregates in the G-CSF formulated sample is by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis, when done under non-reducing conditions can effectively discern the presence of multimeric forms of the protein produced by the process of aggregation.
The stability of the formulation to aggregation or degradation on storage is alternately determined and quantitated by the method of size exclusion HPLC (SE-HPLC) and reverse phase HPLC (RP-HPLC) analysis. In the RP-HPLC analysis, a C-18 RP-column is connected to the HPLC system. The mobile phase used is a triflouroacetic acid / acetonitrile solvent system and the wavelength of detection is set at 280 nm. To the equilibrated column, formulated G-CSF solutions, held at different storage conditions, are injected and the number of peaks obtained and their retention times are noted and compared with the peak area and RT of an external reference standard. The SE-HPLC column is equilibrated using phosphate buffer in the pH range of 2.0 to 4.0 and preconditioned using an aggregated G-CSF solution. To this column, the stability test samples are injected to indicate the presence and quantity of aggregated protein.
Changes in potency during storage are determined in an in vitro cell proliferation based bioassay on a G-CSF responsive cell line like NFS-60. The potency of the sample is determined by comparison of the dose response curve of the test samples with that of a reference standard by parallel line analysis of data.
EXAMPLE 3
This example relates to the stability of different formulations when held at defined storage temperatures (25±2 °C and 5±3 °C). G-CSF was formulated at different concentrations of the protein, in the range of 0.2 to 0.8 mg/ml, in the pH range of 4.1 to 4.8 in buffers with conductivities in the range of l.lmilliSiemens to 2.0 milliSiemens. The presence of different sugar alcohols like sorbitol or mannitol were also tested along with the addition of amino acids like methionine in the final buffers.
Values obtained with select samples from each type of formulation are indicated in tables 1- 5 below, to give a summarized idea of the variation in the stability indicating parameters in all the classes.

The percentage of aggregates in the sample was determined by SE-HPLC analysis. A SE-HPLC column, TSK G 3000 SW XL , 5 urn and 7.8 x 300 mm dimensions was run at 1.0 ml/min flow rate with lOOmM P04 buffer of pH 2.5. The protein peak was detected at 280nm wavelength and quantified using the peak area calculation with respect to a known quantity of reference standard. The tables below will indicate that the percentage of aggregates in the sample is within limits at different conditions of protein concentration, buffer pH, conductivity and presence of methionine. The acceptable limit is