FORM 2 THE PATENTS ACT 1970
(39 OF 1970)
COMPLETE SPECIFICATION
(SECTION 10)
METHOD FOR PREPARATION OF INSULIN LISPRO
CRYSTALS
UNICHEM LABORATORIES LIMITED, A COMPANY
REGISTERED UNDER THE INDIAN COMPANY ACT,
1956, HAVING ITS REGISTERED OFFICE LOCATED AT
UNICHEM BHAVAN, PRABHAT ESTATE OFF. S. V.
ROAD, JOGESHWARI (W) MUMBAI400102
MAHARASHTRA, INDIA
The following specification particularly describes the invention and the manner in which it
is to be performed.

METHOD FOR PREPARATION OF INSULIN LISPRO CRYSTALS
Technical Field of the Invention:
The present invention relates to the method of preparing Insulin Lispro crystals of pharmacopoeial grade.
Background of the Invention:
Insulin Lispro is a fast acting insulin analog. It was first approved for use in the United States in 1996 marketed by Eli Lilly and Company as Humalog, making it the first insulin analog to enter the market. Engineered through recombinant DNA technology, the penultimate lysine and proline residues on the C-terminal end of the B-chain of Insulin are reversed. This modification does not alter receptor binding, but blocks the formation of insulin dimers and hexamers. This allowed larger amounts of active monomeric insulin to be immediately available for postprandial injections. Insulin Lispro has one primary advantage over regular insulin for postprandial glucose control. It has a shortened delay of onset, allowing slightly more flexibility than regular insulin, which requires a longer waiting period before starting a meal after injection.
Human Insulin aggregates into stable hexameric crystals in the presence of zinc. The rate-limiting step in the absorption of the Human Insulin is its dissociation into dimers and monomers when injected in the body. The dissociation causes 30-45 minutes delay in onset of action after administration. However in Insulin LisPro the dimeric interaction is weakened due to reversal of lysine and proline at positions 28 and 29 of the B-chain. In the presence of zinc and phenol, Insulin LisPro forms a stable hexameric crystal form which after injection dissociates into monomers more quickly than human insulin, resulting in a more rapid onset of action.
Typically insulin crystallization process involves preparation of approximately 2 g/L of insulin solution in acidic medium containing organic acid and zinc. The pH of the solution is then adjusted to the isoelectric point of insulin (pH 5.5 to 6.0). The crystals

of Insulin are formed at isoelectric point. In case of Insulin LisPro, however, approximately 0.2% of phenol has to be added for it to be crystallized. The phenolic compound acts as a stabilizer.
US5504188 provides a process to prepare crystalline insulin Lispro, which comprises: crystallizing insulin Lispro from solution comprising insulin Lispro, zinc, at least 0.3N of an organic acid selected from the group consisting acetic, citric, or glycine, and a phenolic at a pH of about 5.5 to about 6.5.
US5952297 describes preparation of monomeric, rhombohedral Insulin Lispro crystals by precipitating hexameric crystals from a solution consisting essentially of insulin Lispro, Sodium chloride, physiologically acceptable buffer and a molar excess of zinc ions at the isoelectric pH of Insulin Lispro. The crystals thus formed do not have tendency to undergo zinc-induced self association. The formulation of monomeric crystals of LisPro prepared according to this process, has a protracted effect and shows extended duration of action.
US5597893 provides a process for preparing a crystalline insulin Lispro alkali metal or ammonium salt, which comprises adjusting the pH of a solution from a pH below the isoelectric point of insulin Lispro to between about pH 8.5 to 9.5 with an alkali metal or ammonium base. The process specifically forms monomeric salt of insulin Lispro.
WO 1998042749 Al discloses Zinc free insulin Lispro crystals having a diameter below 10 urn suitable for pulmonary administration. The crystals are said to have a reduced tendency to associate into aggregates in the dry powder.
Jane Richards et al. in the Journal of Pharmaceutical Sciences (Vol 88 No 9, Sep 1999) disclosed the process to prepare microcrystalline suspension formulation of Insulin LisPro with Ultralente properties, by crystallizing Insulin LisPro in presence of Zinc ions, Sodium acetate and Sodium Chloride while in the absence of phenol.

The process changes the property of the insulin analog by making it slow acting from fast acting Insulin.
Preparation and purification of insulin Lispro involves number of chromatographic stages. In the last stage insulin is polished using reverse phase chromatography. The reverse phase chromatography follows crystallization using Zinc ions near isoelectric point of insulin Lispro. Thus as a general practice before crystallization, insulin Lispro is separated from the mobile phase by lyophilization followed by crystallization using Zinc, centrifugation and drying.
Processes of crystallization reported above need dry insulin Lispro in hand to further recrystallize it. The dry insulin Lispro is dissolved in an acidified aqueous solution and the crystallization is carried out under suitable conditions. Therefore, for these methods the insulin Lispro prepared by any method or isolated from any source is dried by removing the solvent from it and then crystallized under necessary conditions. The process of removing the solvent from the fractions collected from HPLC is cumbersome, lengthy, cost intensive and time consuming process. Also the majority of prior publications listed above disclose the process of crystallization in an aqueous medium.
Prior art processes have some inherent disadvantages or drawbacks. Prior art processes are lengthy. Drying of the crystals to obtain pharmacopoeial grade product is a challenging task. In the reported methods the crystals are freeze dried under reduced pressure. The drying is also carried out under vacuum over dehydrating agents or desiccating agents. The processes are silent on process parameters and do not teach how to perform the process. Prior art processes have multiple steps.
There is need to provide an easy to carry out process, industrially scalable and economic method to prepare recombinant insulin Lispro crystals. Lesser the steps better it is for scale up and industrial use. Lesser steps also mean lesser costs. There is a need to provide optimum drying paramters.

Objects of the invention
The main object of the present invention is to provide a method of preparing pharmacopoeial grade recombinant insulin Lispro crystals in the crystallization solution obtained from reverse phase chromatography during polishing stage, wherein insulin Lispro is not isolated from the crystallization solution obtained from reverse phase chromatography during polishing stage and wherein the crystallization solution contains Organic Solvent.
Another object of the present invention is to develop an easy to carry out, industrially scalable and economic method to prepare recombinant insulin Lispro crystals.
It is also an object of the present invention to reduce the steps involved in the preparation of recombinant insulin Lispro crystals, therefore making the overall process cost effective.
Yet another object of the present invention also aims to develop specific conditions for drying of insulin Lispro crystals to obtain pharmacopoeial grade insulin Lispro.
Yet another object of the invention is to provide a pharmaceutical composition comprising insulin Lispro crystals.
Summary of the Invention:
The present invention provides a method of preparing pharmacopoeial grade recombinant insulin Lispro crystals in the crystallization solution obtained from reverse phase chromatography during polishing stage, wherein insulin Lispro is not isolated from the crystallization solution obtained from reverse phase chromatography during polishing stage and wherein the crystallization solution contains Organic Solvent.
Present invention also provides a method of accelerating process of crystal formation of recombinant insulin Lispro in the crystallization solution obtained from reverse

phase chromatography during polishing stage during scaling up by application of reduced pressure after pH adjustment near isoelectric point at 20-25°C.
Present invention provides a method to prepare pharmacopoeial grade insulin Lispro crystals by drying of the crystallized insulin under vacuum of 5 mbar to 10 mbar at 25°C to 45°C
Present invention also provides pharmaceutical composition comprising recombinant insulin Lispro crystals obtained by the said method.
Present invention provides an easy to carry out, industrially scalable and economic method to prepare recombinant insulin Lispro crystals.
Process to prepare recombinant insulin Lispro crystals has reduced steps and thereby makes the overall process cost effective.
Definition of Terms
The term "crystallization" as used herein refers to the formation of recombinant insulin Lispro crystals.
The term "reverse phase chromatography" is the process of final purification of recombinant insulin Lispro using high performance liquid chromatography with reverse phase column, wherein the reverse phase column is as understood by the person skilled in the art.
The term "polishing stage" is the final stage of purification of insulin Lispro using "reverse phase chromatography".
The term "fractions" refers to the portions of elute collected from "reverse phase chromatography". Fractions contain pure recombinant insulin Lispro.
The term "crystallization solution" refers to the solution obtained by pooling all the "fractions" together.

The term "isoelectric point" is a pH around which net charge of recombinant insulin Lispro is zero wherein recombinant insulin Lispro is precipitated due to low solubility in solution.
"High quantities" of organic solvent is to be interpreted as the percentage of organic solvent from 10% to 35% in the aqueous solution.
The term "scaling up" refers to carrying out the process in the pilot plant or manufacturing plants having capacity more than 12 L and above.
Detailed description of the Invention:
The first embodiment of the present invention relates to a method of preparing pharmacopoeial grade recombinant insulin Lispro crystals in the crystallization solution obtained from reverse phase chromatography during polishing stage, wherein the insulin L ispro is not isolated from the crystallization solution obtained from reverse phase chromatography during polishing stage and wherein the crystallization solution contains Organic Solvent.
According to this embodiment, the eluted fractions of reverse phase chromatography containing pure insulin Lispro are collected and are pooled together to obtain the "crystallization solution".
Crystallization Solution is composed of insulin Lispro in 0.4% acetic acid and at least one Organic Solvent.
The Organic solvent in crystallization solution is a non-aqueous solvent used in the reverse phase chromatography during polishing stage of recombinant insulin Lispro and is selected from acetonitrile, methanol, ethanol and isopropyl alcohol, preferably acetonitrile.
Further the method of preparing pharmacopoeial grade recombinant insulin Lispro crystal comprises the steps of:

a. optionally diluting crystallization solution to obtain crystallization solution
containing lower concentration of recombinant insulin Lispro and lower Organic
solvent concentration;
b. lowering the pH to less than or equal to 3 by acidifying the crystallization solution
obtained in step 'a' using organic acid to obtain acidified crystallization solution;
c. adding liquefied phenol to the acidified crystallization solution obtained in step
'b' to get phenol containing crystallization solution;
d. adding inorganic base to phenol containing crystallization solution obtained in
step 'c' and adjusting the pH to 5.8 - 6.4 to prepare pH adjusted solution;
e. adding metal ion to the pH adjusted solution obtained in step 'd' to prepare metal
ion containing solution;
f. maintaining, while stirring, the metal ion containing solution obtained in step 'e'
initially at 20°C to 30°C for 15 hrs to 48 hrs and then at 2°C to 8°C for 24 hrs to
96 hrs for complete crystallization to obtain recombinant insulin Lispro crystals;
g. separating recombinant insulin Lispro crystals obtained in step 'f from the
solution by filtration to obtain pharmacopoeial grade recombinant insulin Lispro
crystals.
"Crystallization solution" is referred as solution hereafter.
According to this aspect of the invention in step 'a' the solution is optionally diluted either by using the mobile phase used in reverse phase chromatography or using water for injection containing 0.4% acetic acid, to change the concentration of the insulin Lispro as well as organic solvent as desired. The concentration of recombinant insulin Lispro in the crystallization solution is in the range of 1.0 mg/mL to 7 mg/mL. The solution is then treated sequentially, with organic acid in step 'b', and, phenol in step 'c' and metal ion in step 'e' and then it is subjected to crystallization near isoelectric point at said temperature as in step 'f.

The organic solvent in the Crystallization solution after dilution is in the range of 10% to 25%; preferably in the range of 10% to 20% of organic solvent.
Accordingly preparation of pharmacopoeial grade recombinant insulin Lispro crystals from the solution is carried out by lowering the pH of the solution to less than or equal to 3 using organic acid selected from citric acid, acetic acid, lactic acid or oxalic acid; preferably acetic acid as mentioned in step 'b'. The acetic acid stock solution is added to the crystallization solution to obtain acidified crystallization solution in which final concentration of acetic acid is 1.5 - 5.0%, preferably 2.5%. Further according to step c phenol is added to the acidified crystallization solution in an amount of 2ml/L of crystallization solution to obtain phenol containing solution.
The pH of the phenol containing solution is adjusted to pH at 5.8 - 6.4 using a base selected from an inorganic base, preferably selected from Sodium Hydroxide, Potassium Hydroxide, Calcium Hydroxide or Ammonia solution; more preferably Ammonia solution to obtain pH adjusted crystallization solution.
Further according to this embodiment the source of metal ions used to prepare recombinant insulin Lispro crystals from the pH adjusted crystallization solution obtained in step'd' are selected from Zinc ions, Cadmium ions or Cobalt ions. The metal ions are in the form of their halides, acetates or sulphate. Preferably Zinc ions as Zinc Chloride is used. The preferred concentration of Zinc chloride used is from 8 to 24% w/w; more preferably 16% w/w.
According to step 'f of this aspect of invention, the temperature of the metal ion containing solution obtained in step 'e' is maintained at 20°C to 25°C for 15-48 hrs. The pH adjusted solution is gently agitated at this temperature to initiate crystallization. The temperature is then lowered to 2°C to 8°C and maintained for 24 hrs to 96 hrs preferably 72 hrs to complete crystallization of insulin Lispro.

Finally the crystals obtained in step 'f are separated from the solution by the process of filtration or centrifugation to obtain pharmacopoeial grade recombinant Insulin Lispro crystals.
Thus there is provided a method of preparing pharmacopoeial grade recombinant insulin Lispro crystals in the crystallization solution obtained from reverse phase chromatography during polishing stage, wherein insulin Lispro is not isolated from the crystallization solution obtained from reverse phase chromatography during polishing stage and wherein the crystallization solution contains Organic Solvent.
Also the process is shorter and hence cost effective.
The second embodiment of the present invention is a metho d of acceleration of formation of recombinant insulin Lispro crystals during scale up. The method comprises application of reduced pressure at 20-25°C, after pH adjustment near isoelectric point in the crystallization solution obtained from reverse phase chromatography during polishing stage.
The Crystallization Solution is composed of insulin Lispro in 0.4% acetic acid and an Organic Solvent.
The Organic solvent in crystallization solution is a non-aqueous solvent used in the reverse phase chromatography during polishing stage of recombinant insulin Lispro and is selected from acetonitrile, methanol, ethanol and isopropyl alcohol, preferably acetonitrile.
According to this embodiment of the present invention, the method of preparation of pharmacopoeial grade recombinant insulin Lispro crystals described herein is carried out at least on the pilot scale.
According to this aspect the method comprises the steps of:
a. optionally diluting crystallization solution to obtain crystallization solution containing lower concentration of recombinant insulin Lispro and lower organic

solvent concentration;
b. lowering the pH to less than or equal to 3 by acidifying the crystallization solution
obtained in step 'a' using organic acid to obtain acidified crystallization solution;
c. adding liquefied phenol to the acidified crystallization solution obtained in step
'b'to get phenol containing crystallization solution;
d. adding inorganic base to phenol containing crystallization solution obtained in
step 'c' and adjusting the pH to 5.8 - 6.4 to prepare pH adjusted solution;
e. adding metal ion to the pH adjusted solution obtained in step 'd' to prepare metal
ion containing solution;
f. maintaining under reduced pressure, while stirring, the metal ion containing
solution obtained in step 'e' initially at 20°C to 30°C for 15 hrs to 48 hrs and then
at 2°C to 8°C for 24 hrs to 96 hrs for complete crystallization to obtain
recombinant insulin Lispro crystals;
g. separating recombinant insulin Lispro crystals obtained in step 'f from the
solution by filtration to obtain pharmacopoeial grade recombinant insulin Lispro
crystals.
According to this aspect of the invention in step 'a' the solution is optionally diluted either by using the mobile phase used in reverse phase chromatography or using acidified water for injection, to change the concentration of the recombinant insulin Lispro and organic solvent concentration as desired. The concentration of recombinant insulin Lispro in the crystallization solution is in the range of 1.0 mg/mL to 7 mg/mL. The solution is then treated sequentially, with organic acid in step 'b', liquefied phenol in step 'c', metal ion in step 'e' and then it is subjected to crystallization near isoelectric point at said temperature as in step 'f.
The organic solvent in the Crystallization solution after dilution is in the range of 10% to 25%; preferably in the range of 10% to 20% of organic solvent.

Accordingly preparation of pharmacopoeial grade recombinant insulin Lispro crystals from the solution is carried out by lowering the pH of the solution to less than or equal to 3 using organic acid selected from citric acid, acetic acid, lactic acid or oxalic acid; preferably acetic acid as mentioned in step 'b'. The acetic acid stock solution is a dded to the crystallization solution to obtain acidified crystallization solution in which final concentration of acetic acid is 1.5 - 5.0%, preferably 2.5%. Further according to step c phenol is added to the acidified crystallization solution in an amount of 2ml/L of crystallization solution to obtain phenol containing solution.
The pH of the phenol containing solution is adjusted to pH at 5.8 - 6.4 using a base selected from an inorganic base, preferably selected from Sodium Hydroxide, Potassium Hydroxide, Calcium Hydroxide or Ammonia solution; more preferably Ammonia solution to obtain pH adjusted crystallization solution.
Further according to this embodiment the source of metal ions used to prepare recombinant insulin Lispro crystals from the pH adjusted crystallization solution obtained in step'd' are selected from Zinc ions, Cadmium ions or Cobalt ions. The metal ions are in the form of their halides, acetates or sulphate. Preferably Zinc ions as Zinc Chloride is used. The preferred concentration of Zinc chloride used is from 8 to 24% w/w; more preferably 16% w/w.
According to step 'f of this aspect of invention, the temperature of the metal ion containing solution obtained in step 'e' is maintained at 20°C to 25°C for 6-10 hrs. The method of crystallization, in step 'f, is accelerated by applying a constant reduced pressure of 200 to 400 mbar for 15 hrs to 48 hrs. The pH adjusted solution is gently agitated at this temperature and at this pressure to initiate crystallization. The temperature is then lowered to 2°C to 8°C and maintained for 24 hrs to 96 hrs preferably 72 hrs to complete preparation of insulin Lispro crystals. The method is reproducible with uncompromising quality and yield of the product.

Finally the crystals obtained in step 'f were separated from the solution by the process of filtration to obtain pharmacopoeial grade recombinant Lispro crystals.
Thus there is provided a method of accelerating process of crystal formation of recombinant insulin Lispro in the crystallization solution obtained from reverse phase chromatography during polishing stage during scaling up by application of reduced pressure after pH adjustment near isoelectric point at 20-25°C.
Conventionally crystallization of insulin Lispro is carried out after lyophilization, evaporation, repeated concentration and drying of the chromatographic fractions containing pure insulin Lispro. The process is lengthy, time consuming and requires considerable number of unit operations, capital investments and utility requirement. However these steps become necessity as the elute fractions contain organic solvent. Therefore insulin Lispro is separated from solvent containing fractions and a solution of insulin Lispro is prepared to initiate nucleation followed by crystal growth. However when inventor's of present invention carried out crystallization in the solvent containing fractions, surprisingly it was found that even at low concentrations of insulin Lispro, nucleation took place. Slight adjustment of pH around isoelectric point led to crystal growth. Thus inventors could save upon the time, unit operations, capital investments and utility requirement necessary for lyophilization, evaporation, repeated concentration and drying of the fractions.
The inventive aspect of the present invention resides in direct preparation of insulin Lispro crystals in the fractions collected from reverse phase chromatography, in presence of high amount of organic solvent. There is no prior art teaching wherein the preparation of insulin Lispro crystals is reported in a solvent system at such a low concentration of insulin lispro.
Yet another embodiment of the present invention provides a method to prepare pharmacopoeial grade insulin Lispro crystals by drying the crystals under vacuum of 5 mbar to 10 mbar at 25°C to 45°C.

According to this embodiment of the invention, insulin Lispro crystals obtained after filtration/centrifugation stage are dried at 5 mbar to 10 mbar of vacuum at 35-45 DC for 16-24 hrs to give a stable solid form of insulin Lispro suitable for holding and dispensing to fill/finish operations.
Both the parameters viz temperature and vacuum were found to be critical during drying to obtain pharmacopoeial grade insulin Lispro crystals. Higher temperature and higher vacuum could degrade the protein, whereas lower temperature and lower pressure yield insulin Lispro with residual solvents or moisture content above pharmacopoeial limit. When the crystals were dried at 20 mbar pressure and at 32°C temperature, insulin Lispro crystals obtained had 15882 ppm of acetone and 26.3 ppm of acetonitrile with 5.82% moisture content (Estimated as loss on drying). The crystalline insulin Lispro when dried under 5 mbar to 10 mbar of vacuum at 35-45 DC preferably 38°C for 16-24 hrs could yield pharmacopoeial grade insulin Lispro having 60.5 ppm of acetone and acetonitrile below quantification limit.
Thus are provided the specific conditions for drying of insulin Lispro crystals to obtain pharmacopoeial grade insulin Lispro.
Yet another embodiment of the invention is a pharmaceutical composition comprising insulin Lispro crystals prepared by the method described herein.
According to this embodiment, the pharmaceutical composition comprises insulin Lispro crystals obtained by the method described herein and pharmaceutically acceptable excepients.
Further according to this embodiment the pharmaceutical composition is administered intravenously or subcutaneously and the said pharmaceutical excepients are selected from the excepients already known in the art for such compositions, of which any person skilled in the art is well aware.
Recombinant insulin Lispro used for crystallization in this invention was prepared by expressing the precursor molecule Preproinsulin Lispro in E.coli. The Proinsulin

Lispro was prepared from the said Preproinsulin Lispro by solubilization of inclusion bodies followed by clarification, concentration and chemical digestion. Further Proinsulin Lispro was captured and purified in inactive form and was refolded to its native form to yield refolded protein. Refolded protein was subjected to salt precipitation and was captured and purified by hydrophobic interaction chromatography to obtain purified refolded protein. The purified refolded protein was enzymatically cleaved with trypsin and carboxypeptidase B together to remove the C-Peptide to form insulin molecule and the cleaved reaction mixture was purified by ion exchange chromatography to yield purified cleaved protein. The insulin Lispro molecule was further purified in polishing stage by reverse phase chromatography where the reverse phase is C8 and the mobile phase is acetonitrile in water containing 0.4% acetic acid. Protein is eluted in linear gradient and fractionated and pooled to get the insulin Lispro with desired purity.
The following examples are provided merely to further illustrate the preparation of insulin Lispro crystals and they do not limit the invention in any manner.
Example 1: Crystallization at around 15% acetonitrile at 2.0mg/ml concentration.
50 mg of polished pooled sample at 3.7 mg/mL was taken and diluted with 0.4% acetic acid in water and 15% acetonitrile containing 0.4% acetic acid. Final protein concentration of 2 mg/ml and 25ml volume was taken up for reaction. 620uJ of acetic acid was added to lower the pH of the pooled sample to 3 and incubated it for 10 min. 50[il of liquefied phenol was added followed by pH adjustment to 6.14 with ammonia. 40uL of 20% w/v of zinc chloride solution was added. Crystals were observed after 7 hrs of stirring at 25°C.
Example 2: Crystallization at 2.0 mg/ml of insulin Lispro concentration in 10% acetonitrile:

1404 mg of polished pooled sample at 7.8 mg/mL was diluted to 2.0 mg/mL with water containing 0.4% acetic acid and then the resulting solution was further diluted with 10% acetonitrile solution to achieve the final protein concentration to 2.0 mg/ml. Final volume for crystallization obtained was 702 ml. 17.4 mL of acetic acid was added to lower the pH to 3 and incubated for 5 min. 1.4ml of liquefied phenol was added and further kept for stirring for 5 minutes. pH of the crystallization solution was adjusted to 6.03 with ammonia solution and kept under slow stirring. 1.13ml of 20% w/v of zinc chloride solution was added. Well defined crystals were observed after 10 hour.
Example 3: Crystallization at 1.0 mg/ml of insulin Lispro concentration in 10% acetonitrile:
10 mg of polished pooled sample at 4.6 mg/mL was diluted to 1.0 mg/mL with water containing 0.4% acetic acid and 10% acetonitrile containing 0.4% acetic acid to achieve final protein concentration of lmg/ml in 10% acetonitrile Final volume for crystallization obtained was 10 ml. 0.248 mL of acetic acid was added to lower the pH to 3 and incubated for 5 min. 20ul of liquefied phenol was added and further kept for stirring for 5 minutes. pH of the crystallization solution was adjusted to 6.2 with ammonia solution and kept under slow stirring. 8|ul of 20% w/v of zinc chloride solution was added. Well defined crystals were observed after 5 hour.
Example 4: Crystallization in more than 20% acetonitrile at 7.0 mg/mL protein concentration
50 mg of lyophilized insulin Lispro powder was suspended in 22.5% of acetonitrile containing 0.4% acetic acid solution at 7.0 mg/mL. 182|il of 2M Citric acid was added to lower the pH to 3 and incubated for 10 min. 14uJ of liquefied phenol was added followed by the addition of 40|il of 20% Zinc Chloride solution. pH of the reaction mixture was adjusted to 6.18 by ammonia solution and kept under slow stirring. Crystals were observed after 6 hrs of agitation at 25°C.

Example 5: Illustrates the crystallization on pilot scale
26.5 gms of polished pooled sample at 6.44 gm/L was taken and diluted with 9.12L of WFI containing 0.4% acetic acid to a final concentration of 2 g/L. 330 mL of acetic acid was added to lower the pH to around 3 and incubated for 10 min. 26.5ml of liquefied phenol was added followed by pH adjustment to 6.08 with ammonia solution. 22 mL of 20% w/v of zinc chloride solution was added and kept under slow stirring with a constant reduced pressure of 300 mbar for 20.0 hrs at 25°C. After 94hrs vacuum was released and temperature lowered to 2-8°C. Crystals were recovered by membrane filtration under vacuum and washed with 6.0L of chilled WFI, followed by 0.6 L of acetone. The crystals were collected in a Petri plate and dried at 38°C for 24 hr under vacuum of 5 mbar. 25.6 gm of crystals were obtained after drying and were analyzed as per Indian pharmacopoeia. The powder showed 98.84% Insulin Lispro purity, 1.0% other impurities, 0.16% A-21 Des-amido impurity and potency as 27.7 lU/mg.
Example 7: Drying of Crystalline Insulin Lispro
31.7 gms of polished pooled sample at 7.47 g/L was taken and diluted with 11.63 L of WFI containing 0.4% acetic acid to a final concentration of 2 g/L. 397 mL of acetic acid was added to lower the pH to around 3 and incubated for 10 min. 32 ml of liquefied phenol was added followed by pH adjustment to 6.10 with ammonia. 25.4 mL of 20% w/v of zinc chloride solution was added and kept under slow stirring with a constant reduced pressure of 300 mbar for 19 hrs at 25°C. Vacuum was released and the temperature was lowered to 2-8°C for 90 hrs. Crystals were recovered by membrane filtration under vacuum and washed with 8.0 L of chilled WFI, followed by 0.56L of acetone. The crystals were collected in a Petri plate and dried at 38°C for 22 hr under vacuum of 5 mbar. 30.8 gms of crystals were obtained after drying and were analyzed as per Indian pharmacopoeia. The powder showed 98.8% Insulin Lispro purity, 1.06% other impurities, 0.18% A-21 Des-amido impurity and potency

as 27.5 IU/mg. Moisture content (estimated as Loss on drying) in the final drug substance was 6.86%, which is within pharmacopoeial limit (Not more then 10%)
Example 8: Process to prepare the composition comprising crystallized insulin Lispro
300ml of water for injection (WF1) was transferred to a container and 48 g of glycerol was added to the WFI and stirred for 10 mins. 9.45 g of m-cresol was then added to the above solution and kept for stirring (Solution A).
1800 mL WFI was taken in separate container and 5.64 g of sodium phosphate heptahydrate was added to it and kept for stirring for 10 mins followed by pH adjustment to 2.0 using 10% HC1 solution. 11.98 g of pharmacopoeial grade Insulin Lispro crystals were then added to the pH adjusted solution and kept for stirring for 10 mins to obtain a clear solution. 8.8 mL of zinc oxide solution from 0.1% stock was added to the solution and kept for stirring. Solution A was then added to the Insulin Lispro containing solution slowly avoiding frothing. pH of the final solution was then adjusted to 7.2 using 10% sodium hydroxide solution. Final volume was made up to 3 liters with WFI followed by sterile filtration and aseptic filling in USP type - I 10 ml glass vials/ 3 ml cartridges, stoperred and sealed appropriately.

Claims:
We claim,
1. A method of preparing pharmacopoeial grade recombinant insulin Lispro crystals, wherein insulin Lispro is not isolated from the crystallization solution obtained from reverse phase chromatography during polishing stage and wherein the crystallization solution contains Organic Solvent.
2. The method as claimed in claim 1, wherein the organic solvent is selected from acetonitrile, methanol, ethanol and isopropyl alcohol, preferably acetonitrile.
3. The method as claimed in claim 1, comprises the steps of:
a. optionally diluting crystallization solution to obtain solution containing
lower concentration of recombinant insulin Lispro and organic solvent;
b. lowering the pH to less than or equal to 3 by acidifying the crystallization
solution obtained in step 'a' using organic acid to obtain acidified
crystallization solution;
c. adding liquefied phenol to the acidified crystallization solution obtained in
step 'b' to get-phenol containing crystallization solution;
d. adding inorganic base to phenol containing crystallization solution
obtained in step 'c' and adjusting the pH to 5.8 - 6.4 to prepare pH
adjusted solution;
e. adding metal ion to the pH adjusted solution obtained in step 'd' to
prepare metal ion containing solution;
f. maintaining, while stirring, the metal ion containing solution obtained in
step 'e' initially at 20°C to 30°C for 15 hrs to 48 hrs and then at 2°C to 8°C
for 24 hrs to 96 hrs for complete crystallization to obtain recombinant
insulin Lispro crystals;

g. separating recombinant insulin Lispro crystals obtained in step 'f from the solution by filtration to obtain pharmacopoeial grade recombinant insulin Lispro crystals.
4. The method as claimed in claim 5a, wherein the crystallization solution is diluted using either mobile phase of reverse phase chromatography or using acidified water for injection.
5. The method as claimed in claim 5a, wherein concentration of recombinant insulin Lispro is in the range of 1.0 mg/mL to 7 mg/mL.
6. The method as claimed in claim 5a, wherein the concentration of Organic solvent is in the range of 10% to 25%; preferably 10% to 20%.
7. The method as claimed in claim 5b, wherein organic acid is selected from citric acid, acetic acid, lactic acid, oxalic acid; preferably acetic acid, wherein the final concentration of acetic acid in the crystallization solution is 1.5% -5.0% preferably 2.5%.
8. The method as claimed in claim 5c, wherein the liquefied phenol added is in amount of 2ml/L of crystallization solution.
9. The method as claimed in claim 5d, the inorganic base is selected from Sodium Hydroxide, Potassium Hydroxide Calcium Hydroxide or Ammonia solution; more preferably ammonia solution.
10. The method as claimed in claim 5e, wherein the metal ion is selected from Zinc ions, Cadmium ions or Cobalt ions; in the form of their halides, acetates or sulphates; preferably Zinc ion as Zinc Chloride.
11. The method as claimed in claim 10, wherein the concentration of Zinc Chloride is in the range of 8% to 24% w/w; preferably 16% w/w.
12. A method of accelerating process of crystal formation of recombinant insulin Lispro during scaling up, in the crystallization solution obtained from reverse

phase chromatography during polishing stage, wherein the method comprises application of reduced pressure after pH adjustment near isoelectric point.
13. The method as claimed in claim 12, wherein the crystallization solution contains organic solvent selected from acetonitrile, methanol, ethanol and isopropyl alcohol, preferably acetonitrile.
14. The method as claimed in claim 12, comprises the steps of:
a. optionally diluting crystallization solution to obtain crystallization
solution containing lower concentration of recombinant insulin Lispro and
organic solvent;
b. lowering the pH to less than or equal to 3 by acidifying the crystallization
solution obtained in step 'a' using organic acid to obtain acidified
crystallization solution;
c. adding liquefied phenol to the acidified crystallization solution obtained in
step 'b' to get phenol containing crystallization solution;
d. adding inorganic base to phenol containing crystallization solution
obtained in step 'c' and adjusting the pH to 5.8 - 6.4 to prepare pH
adjusted solution;
e. adding metal ion to the pH adjusted solution obtained in step 'd' to
prepare metal ion containing solution;
f. maintaining under reduced pressure, while stirring, the metal ion
containing solution obtained in step 'e' initially at 20°C to 30°C for 15 hrs
to 48 hrs and then at 2°C to 8°C for 24 hrs to 96 hrs for complete
crystallization to obtain recombinant insulin Lispro crystals;
g. separating recombinant insulin Lispro crystals obtained in step 'f from
the solution by filtration to obtain pharmacopoeial grade recombinant
insulin Lispro crystals.

15. The method as claimed in claim 14a, wherein the crystallization solution is diluted using either mobile phase of reverse phase chromatography or using acidified water for injection.
16. The method as claimed in claim 14a, wherein concentration of recombinant insulin Lispro is in the range of 1.0 mg/mL to 7 mg/mL.
17. The method as claimed in claim 14a, wherein the concentration of Organic solvent is in the range of 10% to 25%; preferably 10% to 20%.
18. The method as claimed in claim 14b, wherein organic acid is selected from citric acid, acetic acid, lactic acid, oxalic acid; preferably acetic acid, wherein the final concentration of acetic acid in the crystallization solution is 1.5% -5.0% preferably 2.5%.
19. The method as claimed in claim 14c, wherein the liquefied phenol added is in amount of 2ml/L of crystallization solution.
20. The method as claimed in claim 14d, the inorganic base is selected from Sodium Hydroxide, Potassium Hydroxide Calcium Hydroxide or Ammonia solution; more preferably ammonia solution.
21. The method as claimed in claim 14e, wherein the metal ion is selected from Zinc ions, Cadmium ions or Cobalt ions; in the form of their halides, acetates or sulphates; preferably Zinc ion as Zinc Chloride.
22. The method as claimed in claim 21, wherein the concentration of Zinc Chloride is in the range of 8% to 24% w/w; preferably 16% w/w.
23. The method as claimed in claim 14f, wherein the pressure is preferably reduced to 200 to 400 mbar for 15 hrs to 48 hrs.
24. A method of preparation of pharmacopoeial grade insulin Lispro crystals, wherein the method comprises drying insulin crystals for 16 to 24 hrs under vacuum of 5 mbar to 10 mbar at 25°C to 45°C.

25. The method as claimed in claim 24, wherein drying is preferably carried out under vacuum of 5 mbar to 10 mbar at 35°C to 45°C more preferably 38°C.
26. The . pharmaceutical composition comprising crystallized insulin Lispro obtained by the method as claimed in any preceding claims.