FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
COMPOSITION OF RECONSTITUTION MEDIUM FOR PROTEIN FORMULATIONS
Intas Biopharmaceuticals Limited
An Indian company having its registered office at:
Plot No: 423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, Tal.: Sanand
Ahmedabad-382 210
Gujarat, India
The following specification describes the invention.

FIELD OF THE INVENTION
This invention provides compositions for reconstitution medium of freeze-dried formulations and more particularly provides a composition and method for reconstitution of freeze-dried formulations comprising proteins.
BACKGROUND OF THE INVENTION
Various natural and recombinant proteins have pharmaceutical utility. Once they have purified, separated and formulated, they can be parenterally administered for various therapeutic indications. However, parenterally administered proteins may be immunogenic, may be relatively water insoluble and may have a short pharmacological half life. Consequently, it can be difficult to achieve therapeutically useful blood levels of the proteins in patients.
These problems can be overcome by conjugating the proteins to polymers, such as polyethylene glycol. PEG and other polymer can be conjugated to recombinant proteins to reduce immunogenicity and increase half-life. While preparation of protein-polymer conjugates is beneficial, they cannot be used in a practical manner unless they can be stored for an extended period of time during manufacturing, storage and distribution. Some protein-polymer conjugates, however, rapidly deteriorate even in frozen solutions. To overcome the stability problem of proteins in aqueous formulations, recombinant protein products are stabilized via lyophilization (freeze-drying).
Indian patent no. 207233 discloses a lyophilized powder of an aqueous formulation of PEG-interferon alpha conjugates. The formulations protect PEG-interferon alpha conjugates from loss and degradation during the lyophilization process, as well as degradation during subsequent storage. The formulation also protects loss of biological activity and changes in the degree and/or nature of conjugation during subsequent storage. A preferred PEG-interferon alpha conjugate protectable in the formulations is an interferon alpha-2b-polyethylene glycol (12,000) conjugate.
Lyophilized products require an additional preparation step prior to administration. This process, known as reconstitution, entails mixing the dry drug with a liquid to create an injectable solution, The reconstitution buffer is generally provided along with the lyophilized drug by the manufacturer. However, unique handling procedures need to be followed to avoid physical instability, as the reconstitution involves agitation, formation of foam and froth. After reconstitution some pegylated proteins are not stable due to aggregation and lose its biological

activity due to depegylation. So, the reconstitution medium given along with the lyophilized protein should be able to prevent these instabilities of the polymer conjugated proteins after reconstitution.
US20090062205 discloses compositions useful for reconstitution of concentrated formulations containing protein/peptide pharmaceuticals are provided. The composition generally includes one or more lipids, as well as one or more alcohols that promote and stabilize the formation of (a) lipid molecular assemblies with greater protein encapsulation; (b) protein-lipid complexes and (c) protein and lipid solutions. The reconstitution medium improves the protein-lipid association that in turn alters the pharmaceutical properties.
Liu, Wei., et al., (AAPS Pharmscitech. 2005, vol. 6(2), El 50 - El57) studied about the effect of sucrose-glycine excipient systems on the stability of proteins during lyophilization. Recovery of protein activity after freeze-drying was examined for the proteins in a sucrose-glycine based excipient system in which the formulation was systematically varied. In a sucrose only excipient system, activity recovery of proteins is about 80% and is independent of sucrose concentration over a range from 1 to 40mg/ml. When both sucrose and glycine are used and the ratio of the 2 excipients is varied, however, activity recovery decreases in a pattern that is consistent with the inhibition of activity recovery by glycine crystals, despite the presence of an adequate amount of sucrose to afford protection. Addition of 0.01% polysorbate 80 to the formulation resulted in complete recovery of activity, irrespective of the sucrose-glycine ratio or annealing. Addition of the same concentration of polysorbate 80 to the reconstitution medium caused an increase in activity recovery for each formulation, but the overall pattern remained unchanged.
Jones, Latoya S., et al., (J Pharm Sci. 2001, vol. 90, no. 10, page no. 1466 - 1477) studied an anti-L-selectin antibody as a model protein to investigate the effects of sucrose and/or Tween 20 on protein stability during lyophilization and reconstitution. Native anti-L-selectin secondary structure is substantially retained during lyophilization in the presence of sucrose (1 or 0.125%). However, aggregation of the protein during reconstitution of lyophilized protein powders prepared without sucrose is not reduced by the presence of sucrose in the reconstitution medium. Aggregate formation upon reconstitution is completely inhibited by freeze drying the protein with sucrose and reconstituting with a 0.1% Tween 20 solution. Tween 20 (0.1%) also partially inhibits loss of native anti-L-selectin secondary structure during lyophilization. However, upon reconstitution the formulations lyophilized with Tween 20 contain the highest levels of aggregates. The presence of Tween in only the reconstitution solution appears to inhibit the

transition from dimers to higher order oligomers. Potential mechanism(s) for the Tween 20 effects were investigated. However, no evidence of thermodynamic stabilization of anti-L-selectin conformation (e.g., by Tween 20 binding) could be detected.
Mei Z. Zhang et al., (Pharm Res. vol. 12, no. 10; 1995, page no. 1447 - 1452) optimized the effect on aggregate formation using recombinant keratinocyte growth factor (KGF). The protein was lyophilized under suboptimal conditions to induce aggregation and precipitation upon reconstitution with water. A series of additives were examined by UV spectrophotometry and size exclusion chromatography (SEC-HPLC) for their effects on decreasing the degree of KGF aggregation and precipitation by the increase in recovery of soluble monomer. Several additives resulted in a significant reduction of aggregation, including sulfated polysaccharides, surfactants, polyphosphates, and amino acids. A similar effect was achieved by adjusting the ionic strength of the reconstitution medium. SEC-HPLC indicated that the amount of soluble monomer was also increased by these additives suggesting that the recovery of the soluble protein correlates with the native, monomeric protein. The results suggest that optimization of reconstitution conditions will be a useful methodology for increasing the recovery of soluble, active proteins and that for KGF, the recovery of the soluble protein correlates with the native, monomeric form.
MeiZ.Zhang et al, (Pharm Res. vol. 13, no. 4, 1995, page no. 643 - 646) studied that the inclusion of additives in the reconstitution medium such as polysaccharides, polyphosphates, and amino acids reduces aggregation and also enhanced recovery of native, active protein. They used ribonuclease A (RNAse A) and interleukin-2 as a model protein for testing various additives in their ability to reduce aggregation upon lyophilization and reconstitution.
As it can be seen from the prior art, there is an urgent need to develop the composition of reconstitution medium which increases the stability and reduces "or prevents aggregation, depegylation for lyophilized protein products especially lyophilized pegylated protein products after reconstitution.
SUMMARY OF THE INVENTION
The invention relates to the compositions for preparing the reconstitution solution for lyophilized protein formulations comprising amino acids, sugar alcohols either alone or in combination.
In one aspect of the invention wherein the reconstitution solution further comprising surfactants selected from the group consisting of polysorbate or poloxamer either alone or in combination.

In the second aspect of the invention wherein the reconstitution solution further comprising tonicity modifiers selected from the group consisting of NaCl or suitable salts therewith.
In yet aspect of the invention wherein the reconstitution solution further comprising stabilizers selected from the group consisting of benzyl alcohol, m-cresol.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the stability profile obtained by using SEC-HPLC. In that chromatogram, Peak 1 shows the aggregates formed when using WFI as a reconstitution medium, Peak 2 shows the MonoPEG obtained using said composition as a reconstitution medium (red line) and WFI as a reconstitution medium (blue line).
By using WFI (Blue line) as a reconstitution solution, the aggregates (Peak 1) and free interferon (Peak 3) were found to be more which in turn decrease the percentage of monoPEG (Peak 2) present in the reconstituted formulation,
At the same time by using the said reconstitution composition of the present invention (Red line), there are no aggregates and the percentage of monoPEG (Peak 2) present in the reconstituted formulation is high which in turn shows the high stability.
DESCRIPTION OF THE INVENTION
The present invention is directed to the compositions of media useful for reconstitution of lyophilized protein formulations. The compositions can be used with lyophiiized protein formulations comprising protein to form stabilized protein solutions.
The invention relates to the compositions for preparing the reconstitution solution for dried protein formulations, comprising amino acids, surfactants, tonicity modifiers, sugars either alone or in a suitable combination.
Sugar alcohols, primary alcohols and secondary alcohols are frequently added as excipients in the formulation of proteins to improve stability and solubility. In case of lyophilized protein formulations the primary and secondary alcohols cannot be used due to the technical restrictions associated with lyophilization process. Hence due to these limitations an alternative approach is

tried by using the said excipients in lyophilized formulations is explored in this invention. The present invention demonstrates that primary, secondary and sugar alcohols can be used as components in the reconstitution medium for lyophilized protein formulations. It has been discovered that concentrations of the above alcohols which are below the effective concentration have the effect of structurally stabilizing the intended proteins without greatly impeding their function. When the compositions are to be used to reconstitute lyophilized protein formulations intended for administration to humans or animals, the alcohols should be such that they can be safely administered. Such alcohols may include benzyl alcohol, m-cresol, ethanol, glycerol and polyethylene glycol (PEG). Similarly Sugar alcohols which can be used, mannitol, arabitol, sorbitol and xylitol have been shown to have some stabilizing effect on protein lyophilized formulations after reconstitution.
Generally amino acids are used in the pharmaceutical formulations for enhancing the stability of the intended molecules. In the present invention the use of amino acids as a component in the reconstitution medium in order to prevent or control the depegylation rate of the lyophilized PEGylated protein formulation is explored. It is found that use of amino acids such as glycine, lysine, cysteine, histidine, histidine hydrochloride, arginine and glutamine either alone or in combination in the reconstituted solution prevents or controls the depegylation rate of the PEGylated molecule significantly up to certain period of time which will enhance the stability of the PEGylated lyophilized protein formulation after reconstitution. This ensures the safety and efficacy of the molecule upon reconstitution, if stored for considerable period of time before administration to the patients in order to fulfill the patient compliance.
In general, surfactants are included in the pharmaceutical formulations for prevention of adsorption of protein or other formulation components to the surface of the primary container and closures. The concentration of surfactants used in pharmaceutical formulations will be better if used well below the critical micellar concentration (CMC) to avoid aggregation in protein formulations. But in most of the formulations, the surfactant is present well above the CMC level which shows better stability of drug molecules. Simultaneously if combination of surfactants is used in formulations it will lead to instability issues especially with the protein formulations. This restricts the use of combination of surfactants in the protein formulations in order to prevent the instability issues. Hence there is a strong need to find an alternative approach to use combination of surfactants in the protein formulations to increase the stability of the molecule in long-term storage. The present invention provides an alternative approach to use one of the surfactant in the lyophilized PEGylated protein molecule and the other in the reconstitution medium for the same.

The use of surfactants in the present invention includes polysorbates such as polysorbate 20, polysorbate 40, polysorbate 80 and poloxamers. The use of combination of surfactants by the above mentioned approach provided better stability both in the lyophilized formulation and upon reconstitution of the same for intended period of time.
In genera], the tonicity modifiers are used in pharmaceutical formulations for maintaining the isotonicity of the solution. In certain cases, the pharmaceutical compositions consists of very less concentration of tonicity modifiers due to certain limitations of useb in large amounts especially in the case of lyophilized formulations and hence the isotonicity 0f the formulation will not be achieved. Hence an alternative approach of addition of isotonicity modifiers in the reconstitution solution in order to obtain the desired isotonicity of the protein formulation. The present invention further reveals that the addition of tonicity modifiers in the formulation composition especially in lyophilized PEGylated protein formulation leads to severe depegylation and aggregation issues in turn decreasing the stability of the formulation. The use of the same tonicity modifiers such as sodium chloride, mannitol, sorbitol, calcium chloride in the reconstitution solution will avoid the above mentioned instabilities thereby maintaining the desired isotonicity of the formulation.
The following example illustrates further the composition of reconstitution medium described in the present invention and the means of carrying out the invention to obtain a stable pharmaceutical formulation of pegylated interferon alpha 2b.
Example 1
PEGylated Recombinant Human Interferon Alpha 2b drug product (50mcg/ 0.5 ml, 80mcg/ 0.5 ml and 150mcg/ 0.5 ml) available as lyophilized cake is reconstituted before injection. An appropriate reconstitution solution is one which (should be physico-chemically compatible with the drug product and provide stability) stabilizes the product after reconstitution for at least 24 hours (as recommended by the Reference Medicinal Product). Here effect of reconstitution solution on purity of drug product as determined by monopeg content and the related impurities present, was studied. Monopeg content of the drug product was determined immediately after reconstitution, approximately 60 minutes after reconstitution and approximately 7 hours after reconstitution.
Pegylated Recombinant Human Interferon Alpha 2b drug product (50mcg/ 0.5 ml, 80mcg/ 0.5 ml and 150mcg/ 0.5 ml) is reconstituted with appropriate volumes 0f reconstitution solution to get

final concentration to 0.1 mg/ml, 0.16 mg/ml and 0.3 mg/ml respectively. At these concentrations, the maximum amount of protein that can be loaded for SE-HPLC column is 10, 16 and 30 μg respectively.
List of composition of the reconstitution solution studied was given below
1) Water for Injection
2) 0.5 % Sodium Chloride
3) 0.5 % Sodium Chloride + 0.1 mg/ml Polysorbate 80
4) 0.5 % Sodium Chloride + Benzyl Alcohol
5) 0.5 % Sodium Chloride + 5mM Glycine + 0.1 mg/ml Polysorbate 80
6) 25 mg/ml Mannitol + 0.1 mg/ml Polysorbate 80
7) 25 mg/ml Sorbitol + 0.1 mg/ml Polysorbate 80
Here, the Drug Product was reconstituted with a predetermined volume of reconstitution solution and a fixed amount of protein was loaded on SEC-HPLC for analysis. The same reconstituted drug product (DP) solution was stored at 2 °C to 8 °C and reanalyzed after a certain time interval.
RESULTS:
1. Reconstitution with WFI

Sample Time (min) Aggregates Dipeg Monopeg Native
Peg-IFN Alfa 2b 0 0.356 2.762 95.846 1.066

320 2.218 3.21 93.593 0.979
This table shows the result contains WFI used to reconstitute PEGylated recombinant human interferon Alfa 2b drug product and the stability of the reconstituted drug product was studied over a period of time. SEC data presented in above Table shows that, PEGylated recombinant human interferon Alfa 2b drug product monopeg content changed with storage period. Higher aggregation was seen in the sample analyzed approximately 6 hours after reconstitution while free Interferon content remained approximately the same. This indicated a modification in the reconstitution medium would be required,

2. Reconstitution with 0.5 % Sodium Chloride

Sample Time (min) Aggregates Dipeg Monopeg Native
Peg-IFN Alfa 2b 0 1.09 2.795 95.372 0.743

-320 5.816 2.839 90.626 0.719
In this combination also the aggregates are increasing with time.
3. Reconstitution with 0.5 % Sodium Chloride+ 0.1 mg/ml Polysorbate 80

Sample Time (min) Aggregates Dipeg Monopeg Native
Peg-IFN Alfa 2 b 0 1.52 3.05 94.06 1.38

40 1.58 3.09 93.87 1.46

420 1.52 2.98 93.78 1.72
In this combination of reconstitution solution, the free interferon (native) is increasing with time. So the ideal reconstitution solution should control the depegylation and aggregates both up to a certain period of time.
4. 0.5 % Sodium Chloride + 5mM Glycine + 0.1 mg/ml Polysorbate 80

Sample min Aggregates Dimer Dipeg Monopeg Native
Peg-IFN Aifa 2 b 0 1.422 0.395 3.064 94.08 1.039

40 1.246 0.404 3.04 94.042 1.267

420 0.946 0.437 3.044 94.356 1.218
This composition of reconstitution solution controls aggregation and depegylation and ultimately increases the stability of the molecule. This combination found to be a working composition for PEG-IFN alfa 2 b lyophilized drug product.

We Claim
1. A reconstitution medium for the reconstitution of lyophilized protein formulations comprising amino acids, sugar alcohols either alone or in combination.
2. A reconstitution medium according to the claim 1 wherein the amino acid is selected from the group consisting of glycine, lysine, cysteine, histidine, histidine hydrochloride, arginine and glutamine either alone or in combination thereof.
3. A reconstitution medium according to the claim 1, wherein the sugar alcohol is selected from the group consisting of mannitol, arabitol, sorbitol and xylitol either alone or in combination thereof.
4. A reconstitution medium according to claim 1 further comprising surfactants selected from the group consisting of polysorbate or poloxamer either alone or in combination.
5. A reconstitution medium according to claim 1 further comprising tonicity modifiers selected from the group consisting of NaCl or suitable salts therewith.
6. A lyophilized protein formulation according to the claim 1, wherein the protein formulation.comprising pegylated interferons.
7. A pegylated interferon according to the claim 5 wherein the pegylated interferon is pegylated interferon alpha 2b.
8. A reconstitution medium according to the claim 1 wherein the composition comprising glycine, polysorbate 80 and sodium chloride.
9. A reconstitution medium according to the claim wherein the osmolality of the said reconstitution composition is ranging from 110 to 190mOsm/L.
10. A method for the reconstitution of pegylated protein preparation to produce a stabilized pegylated protein solution comprising: combining a lyophilized pegyalted protein

formulation and a reconstituted medium to form a reconstituted protein solution wherein the reconstituted formulation has no aggregation and <4% free protein.