FIELD OF INVENTION

The present invention relates to a method of preparation and a frozen therapeutic protein preparation wherein multiple freeze/thaw induced protein dimerization and/ or particulate matter formation is reduced.

BACK GROUND OF THE INVENTION

A major challenge in industrial scale production of therapeutic proteins is their stability on long-term storage. Storage of any aqueous protein preparation renders them susceptible to various physical and chemical degradations resulting in activity loss. Even though freezing of the protein preparation seem to be a plausible approach especially for long-term the freezing process may itself poses significant threats to the protein quality especially if multiple freeze thaw cycles are involved or expected.
Multiple freeze thaw cycles can lead to accumulation of protein dimers or particulate matter. Formation of particulate matter and/or multimerization of the protein can significantly affect protein quality and perturb its pharmacokinetic or pharmacodynamic profiles [http://www.pharmtech.unierlangen.de/news/APV_Malta/Abstract_Straller.pdf]. Therefore it is important to minimize multimerization or formation of particulate matter during multiple freeze thaw cycles. A robust freezing process may additionally aid in circumventing cold chain breach especially during transportation of the frozen preparation.

Excipients such as amino acids sugars surfactants salts polyols antioxidants polymers chelating agents etc have been employed for maintaining protein stability in a pharmaceutical formulation (Hand book of pharmaceutical biotechnology Shayne Cox Gad pg: 399 Edition: 1). Sugars polyhydric alcohols amino acids and surfactants in particular have also been used for preventing freeze/thaw induced protein aggregation. However a generalized approach towards choice of excipients or a freezing process may not be feasible across the different class of proteins.

Polysorbate 80 has been reported to inhibit aggregation of Factor IX and IL-1 during freeze/thaw cycles (Chang BS et al. Journal of pharmalogical Science 12 1996 1325-30). Similarly Polysorbate 20 has been suggested for decreasing insoluble aggregate formation in recombinant Factor-XIII at a protein concentration range from 1-10mg/ml. A molar ratio of 4:1 of Tween20: protein has also been suggested for preventing freeze/thaw induced aggregation in case of recombinant human growth hormone.

Apart from aggregate content in a protein preparation presence of monomeric dimeric or multimeric forms of a protein may also influence the bioactivity of the protein. US5917021 suggests use of a combination of sucrose and histidine/glycine for maintaining more than 85% of a single-chain antigen-binding protein in monomeric form. However greater than just 85% monomer content may not be sufficient for certain proteins to obtain desired activity. Further the teaching is limited for a single chain antigen binding protein and cannot be extended to humanized or chimeric antibodies which are divalent. WO2008101175 has suggested use of sorbitol as a substitute of mannitol and a particular freezing or thawing rate to prevent mannitol induced aggregation during a slow freeze thaw process.

The principal object of the present invention is to provide a process and a therapeutic protein formulation in particular for an antibody which comprises not less than 95% of the protein in monomeric form when subjected to multiple freeze thaw cycles. An additional object of the present invention is to propose suitable concentrations of a sugar surfactant or a combination thereof such that multiple freeze/thaw associated particulate matter could be reduced. An additional object of the invention is to provide a process of freezing and/or thawing a protein preparation such that the resulting preparation exhibit not less than 95% of the protein in monomeric form.

SUMMARY OF THE INVENTION:

The present invention provides a frozen therapeutic protein preparation and a method of preparing the same such that the preparation contains not less than 95% of the protein in monomeric form when subjected to multiple freeze/thaw cycles.

BRIEF DESCRIPTION OF DRAWINGS:

Fig.1: is an illustration of folds decrease in freeze/thaw induced particulate matter observed in formulation 6 when compared with formulation 5 after multiple freeze/thaw cycles described in example 1.

Fig.2: is an illustration of folds decrease in freeze thaw induced dimer content in formulation 2 3 and 4 when compared with formulation 1after multiple freeze/thaw cycles described in example 1.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the disclosed invention encompass a frozen therapeutic protein in particular a divalent antibody preparation which contains not less than 95% of the protein in monomeric form upon multiple freeze/thaw cycles. Multiple freeze- thaw cycles refer to more than one 2 3 4 or up to 5 freeze thaw cycles. In context of the present invention divalent antibody/ies refer to an immunoglobulin molecule containing two heavy chains and two light chains.

In another embodiment of the invention provides a method of preparing a frozen therapeutic protein in particular a divalent antibody preparation which contains not less than about 95% of the protein in monomeric form upon multiple freeze/thaw cycles.

In another embodiment of the invention provides a method of preparation of a frozen therapeutic protein in particular a divalent antibody preparation exhibiting not less than 95% of the protein in monomeric form upon multiple freeze/thaw cycles wherein in the freezing is performed in flexible bags.

In another embodiment the freezing is conducted in a blast freezer which utilizes freezing plates such that the heat dissipation occurs across the largest surface area of the flexible bag.
In another embodiment of the invention the thickness of the freezing bag post filling of the aqueous protein preparation is almost about 4cm.

In another embodiment of the invention the freezing bags are filled maximally up to about 70% of its total fill volume such that the thickeness of the freezing bag is about 4cm.

In yet another embodiment of the invention a process of freezing a protein formulation to a temperature up to about -50?C or below wherein the freezing is conducted in flexible bags of thickness of about 4cm.

In another embodiment of the invention provides the aqueous protein preparation is frozen to a solid state within in a period not extending more than about 120minutes.

In further embodiment of the invention provides storage of the frozen protein preparation at a temperature above -50?C but below the freezing point of the aqueous protein preparation for at least about 24hrs.

In another embodiment the frozen protein preparation is thawed to a final temperature of about 23±2?C without incubation at any temperature between storage temperature and final temperature.

In another embodiment of the invention provides use of a non reducing sugar for preventing freeze thaw associated dimerization of a protein preparation. Some non-limiting examples of non-reducing sugars may include trehalose sucrose lactose maltose etc.

In a further embodiment of the invention the non-reducing sugar is used at a concentration of about 5-7%.

In another embodiment the invention provides method of decreasing freeze thaw associated dimerization up to about 2.5 folds by use of a non-reducing sugar as an excipient for freezing a therapeutic protein preparation in particular an antibody preparation.

In another embodiment the invention provides use of a non-ionic detergent for freezing a therapeutic protein preparation in particular an antibody preparation.

In another embodiment the invention provides the a non-ionic detergent is used at a concentration of about 0.04% to about 0.14%.

In yet another embodiment provides a method of reducing freeze thaw associated particulate formation up to about 9 folds in a protein preparation by use of non-ionic detergent for freezing. Different non-ionic detergents are known in the art some non-limiting examples may include polysorbates NP40 etc.

In another embodiment of the invention provides use of a combination of a non-ionic detergent and a non-reducing sugar for preventing freeze thaw associated dimerization of a monomeric protein preparation in particular an antibody preparation.

In another embodiment the invention provides a frozen therapeutic protein preparation comprising a buffer a non-ionic surfactant and/ or a non reducing sugar such that multiple freeze/ thaw associated dimerization and/or particulate matter formation is reduced. Different buffers are known in the art some non limiting examples may include citrate buffer phosphate buffer acetate buffer etc.

The dimer or particulate content of the preparation could be estimated by many known techniques in the art such as SE-HPLC (size exclusion high performance liquid chromatography) or LBPC (liquid borne particle count).

The invention is more fully understood by reference to the following examples. These examples should not however be construed as limiting the scope of the invention.

EXAMPLES

Exapmle: 1

Compositions of formulations:

One humanized antibody and one mouse human chimeric antibody preparation were utilized for the study and were formulated as described in table 1

Formulation Description

Formulation 1 51 mM Phosphate buffer pH 6.2 humanized antibody (~30mg/ml)

Formulation 2 51 mM Phosphate buffer pH 6.2 60mg/ml trehalose humanized antibody (~30mg/ml)

Formulation 3 51 mM Phosphate buffer pH 6.2 0.04% polysorbate 20 (%w/v) humanized antibody (~30mg/ml)

Formulation 4 51 mM Phosphate buffer pH 6.2 60mg/ml trehalose 0.04% polysorbate 20 (%w/v) humanized antibody (~30mg/ml)

Formulation 5 25 mM Citrate buffer pH 6.5 chimeric antibody (~18mg/ml)

Formulation 6 25 mM Citrate buffer pH 6.5 0.14% polysorbate 80 chimeric antibody (~18mg/ml)

Table 1: Different formulations of proteins used in this study excipients of each formulation are listed.

The formulations listed in table 1 were filled in flexible bags. Fill volume of about 70% (0.63±0.02) of total fill volume yielded a thickness of about 4cm. In case the thickness of the bag was not 4cm multiple bags were stacked to attain the thickness. Filled bags were placed in the blast freezer between two freezing plates ensuring that the heat transfer occurred across the largest available surface area of the bags. Freezing Cycle is elaborated in table 2.

S. No Freezing parameter limit
1 Pre-cooling time (min) 30
2 Cooled plate temperature (?C) Less than -50
3 Freezing plate temperature (operating temperature ?C) Less than -50
4 Freezing cycle time (min) About 113
5 Sample thickness to be maintained (cm) 4

Table 2: Freezing process details

Once frozen the bags containing the frozen protein preparation is stored at -20?C. The frozen protein was at least stored for about 24hrs before thawing. Frozen samples were thawed directly to a final temperature of about 23±2?C. The time required for thaw is volume dependent for instance on an average 7L volume took about 15hrs to thaw to a final temperature of about 23±2?C.

To access role of different excipients and the freeze thaw process multiple freeze/thaw (FT) cycles were followed as per the mentioned parameters.

Particulate matter due to repeated freeze thaw and dimer content of the protein was evaluated by LBPC (liquid borne particle counter) and SE-HPLC (Size exclusion chromatography) respectively.

Use of polysorbate exhibited about 9 fold decrease in freeze thaw associated particulate matter formation in formulation 5 and 6 (Figure 1) up to 5 freeze/thaw cycles.

Size exclusion chromatography was performed to quantitate the monomer content of the protein preparation upon multiple freeze/thaw. Table 3 enumerates monomer content of the formulations at different freeze/thaw cycles. Formulation 1 containing lacking excipients other than buffer exhibited protein monomer content of less than 95% at 3rd and 5th freeze/thaw cycle. Interestingly formulation 2 3 and 4 exhibited more than 95% monomeric form of protein at 3rd freeze thaw cycle. Formulation 2 and 4 preserved more than 95% monomeric protein content even after 5th freeze/thaw cycle.

Figure 2 represents the fold decrease in freeze/thaw associated protein dimerization with respect to formulation 1. It is clear that the formulation 2 and formulation 4 exhibit maximal reduction in freeze/thaw associated protein dimerization. Similarly it could be inferred that formulation 6 exhibit up to about 9 fold reduction in freeze/thaw associated particulate formation with respect to formulation 5 lacking any excipient other than buffer.

Sample % Monomer
Formulation 1 % Monomer
Formulation 2 % Monomer
Formulation 3 % Monomer

Formulation 4

3 FT 94.76 97.43 95.11 97.445
5 FT 94.97 97.98 94.99 98.055

Table 3: Percentage of monomer content in different formulation analyzed by SE-HPLC after 3rd and 5th freeze-thaw cycles.

WE CLAIM

1. A method of preparing a frozen therapeutic protein formulation comprising, a divalent antibody preparation having not less than about 95% of the protein in monomeric form after multiple freeze/thaw cycles, wherein in the freezing is performed in flexible freezing bags.

2. The method of claim 1, wherein the number of multiple freeze/thaw cycles is approximately about 2, 3, 4 or 5.

3. The method of claim 1, wherein the flexible freezing bags are filled maximally up to about 70% of its total fill volume and wherein the thickness of the freezing bags is about 4cm.

4. The method of claim 1, wherein the freezing apparatus used is a blast freezer comprising freezing plates which allows the largest heat dissipation through the surface area of the flexible freezing bags.

5. A method of preparing a frozen therapeutic protein formulation comprising, a divalent antibody preparation having not less than about 95% of the protein in monomeric form after multiple freeze/thaw cycles, wherein in the freezing is performed in flexible bags and wherein the reduced particulate formation is achieved using non-reducing sugar or non-ionic detergent or combination thereof.

6. The method of claim 5, wherein the freeze thaw associated particulate formation is reduced up to about 9 folds and dimerization is reduced up to about 2.5 folds in a protein formulation.

7. The method of claim 5, wherein the concentration of non-reducing sugar is about 5-7%.

8. The method of claim 5, wherein the non-reducing sugar is trehalose, sucrose, lactose or maltose.

9. The method of claim 5, wherein the concentration of non-ionic detergent is about 0.04% to about 0.14%.

10. The method of claim 5, wherein the non-ionic detergent is polysorbate or NP40.