FORM 2
THE PATENTS ACT 1970
(39 OF 1970)
COMPLETE SPECIFICATION (SECTION 10)
METHOD FOR PREPARATION OF INSULIN CRYSTALS
UNICHEM LABORATORIES LIMITED, A COMPANY REGISTERED
UNDER THE COMPANIES ACT, 1956, HAVING ITS REGISTERED OFFICE
LOCATED AT UNICHEM BHAVAN, PRABHAT ESTATE, S. V. ROAD,
JOGESHWARI (WEST), MUMBAI - 400 102, MAHARASTRA,INDIA

METHOD FOR PREPARATION OF INSULIN CRYSTALS
Technical Field of the Invention:
The present invention relates to the method of preparing Insulin crystals of pharmacopoeial grade.
Background of the Invention:
Since the introduction of insulin in 1920, continuous improvements in the processes of preparation of insulin have been made to cope up with the increasing demand of the drug to treat diabetes mellitus. The process of purification of insulin has been studied extensively for more than 50 years. The purification of insulin in the present day includes purification by chromatography followed by crystallization of insulin. The crystallization of insulin is well known and is dated back to 1926 when Abel crystallized insulin in the isoelectric region from a solution buffered with brucine, pyridine, and ammonium acetate (Abel J.J., Proc. Natural acad Sci.U.S12:132 (1926)).
Ideally insulin crystallizes as rhombohedral zinc hexamer in presence of crystallization promoting metal (Zn as ZnC12) in a medium with pH around the isoelectric point of insulin, which is around 5 to 7. There are other crystallization promoting metals such as Cobalt, Nickel, Cadmium, Copper, Manganese and Iron; however Zinc is employed in most cases.
Several processes are reported for the crystallization of insulin. They either improve the overall process of crystallization of insulin or improve the quality of the crystallized insulin. According to GB733740 (1952), the presence of halogen in ionic form, in a concentration over 0.2 mole per liter in the crystallization medium can improve the conditions of insulin crystallization to such a degree that the insulin, irrespective of origin and small amounts of impurities, crystallizes in particularly pure

form and in rhombohedral shape as sharp-edged crystal bodies bounded by plane crystal faces. Crystallization of insulin to produce crystals of predetermined size and quality is reported in US2799622 (1953) and US2819999 (1953), wherein crystallization is carried out by seeding with the insulin crystals and freeze-dried insulin, respectively. WO2008065138 Al (2006) provides a process of crystallization of insulin in the presence of chaotropic agent and US20110070219 Al (2009) discloses crystallization of recombinant proteins under high pressure in presence of polyethylene glycol as a precipitating agent.
Large scale production of highly purified porcine and bovine insulin was reported in WO9000177 (1988), wherein insulin was purified by chromatographic techniques using gels with a high chemical and physical stability and the pure insulin obtained was precipitated using zinc ions.
In all of the above methods dry insulin is dissolved in an acidified aqueous solution and the crystallization is carried out under suitable conditions. Therefore, for these methods the insulin prepared by any method or isolated from any source is dried by removing the solvent from it and then crystallized under necessary conditions.
There are reports of crystallization of other similar molecules for e.g. EP0692489 Bl (1994) reports crystallization of Lys Pro human insulin from solution comprising LysB28ProB29 human insulin, zinc, atleast 0.3 N of an organic acid and a phenolic compound at a pH of about 5.5 to about 6.5; EP2708550 Al (2011) discloses a method of preparing insulin glargine (GlyA21-ArgB31-AryB32-human insulin) crystal, comprising crystallizing the insulin glargine at pH 7.0 - 9.0 in a solution containing a recombinant insulin glargine (2.8 to 3.2 mg/mL), an organic solvent of a 10 - 30% concentration by volume, a zinc compound, a phenol derivative, a salt and an organic acid. Crystallization of insulin aspart to obtain stable crystals has been disclosed in CN103342746 B. The crystallization is done at 10°C to 30°C at pH 6.0 to 6.5, in a solution containing glycine, recombinant insulin aspart (3-10 mg/mL preferably 3-5

mg/mL), 10 - 30% of organic solvent, phenolic compound, salt solution and zinc ions.
Methods of crystallization reported in the three patents listed above are specific to particular molecule and cannot be applied to insulin other than insulin LysB28ProB29, insulin glargine and insulin aspart, respectively. The present inventors tried the same processes with variation as and when required (pH) to apply the same process to recombinant human insulin. The process resulted in the amorphous recombinant human insulin.
With advent of manufacturing of recombinant human insulin and various
*
chromatographic techniques to purify recombinant human insulin the process of purification and crystallization became more and more complex. Preparation and purification of insulin 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. Thus as a general practice before crystallization, insulin is separated from the mobile phase by lyophilization followed by crystallization using Zinc, centrifugation and drying.
Processes of crystallization reported above need dry insulin in hand to further recrystallize it. The process of removing the solvent from the fractions collected from HPLC is a lengthy, cost intensive and time consuming process. Also the majority of prior publications listed above disclose the process of crystallization in an aqueous medium. There are only two reports of crystallization of insulin analogues in the presence of organic solvents. EP2708550 Al reports method of crystallization of insulin glargine and crystallization of insulin aspart is disclosed in CN103342746 B. However as mentioned above both these processes are not useful for the insulin in the present invention.

Further according to the prior art, insulin crystals are dried to finally obtain purified, dried crystalline insulin. Usually insulin crystals are freeze dried under reduced pressure where aqueous suspensions are frozen at a temperature below 0°C on drying sheets and then dried under reduced pressure. CN103512318 A (2012) discloses a process of freeze-drying of insulin, by pre-freezing, primary drying and secondary drying. This is a slow and lengthy process. It is also cost intensive as it requires huge capital investment and more space for lyophilization as also the failure of freezing equipment in large scale would risk a total loss of product.
Other methods used for drying of insulin crystals are vacuum drying over dehydrating agents such as Sulphuric Acid or Phosphorous Pentoxide (US2626228 1945), drying the insulin crystal suspension in a centrifugal dryer using a stream of gas/air (US6408536 1999) or air drying. These methods either involve use of corrosive chemicals or are expensive as these demand specific unit and apparatus.
Albeit US6408536 in its prior art makes a general statement that insulin crystals can be dried by isolating the crystals from a crystal suspension by filtration and drying the filtered cake under reduced pressure at a temperature above 0°C, it is silent on process parameters and does not teach how to perform the process. Prior art also does not teach the drying process to produce pharmacopoeial grade insulin. Present invention is related to process of preparing and drying insulin crystals to produce pharmacopoeial grade insulin. In other words US6408536 and the prior art sited in it do not teach the specific conditions required to dry insulin crystals under vacuum and to yield pharmacopoeial grade insulin.
The main object of the present invention is to develop an easy to carry out, industrially scalable and economic method to prepare crystalline insulin. It is also an object of the present invention to reduce the steps involved in the preparation of crystalline insulin, therefore making the overall process of preparing insulin, cost effective. The present invention also aims to develop specific conditions for drying of insulin crystals to obtain pharmacopoeial grade insulin.

1
Summary of the Invention:
The main object of the present invention is to develop a method of crystallizing insulin in the crystallization solution obtained from reverse phase chromatography during polishing stage, wherein the insulin is not isolated from the crystallization solution obtained from reverse phase chromatography during polishing stage.
In yet another object of the present invention, insulin is crystallized in the crystallization solution obtained from reverse phase chromatography during polishing stage, wherein the crystallization solution comprises high quantities of organic solvent and wherein the concentration of insulin in the crystallization solution is in the range of 1.0 mg/mL to 10 mg/mL.
Yet another object of the present invention is to develop a method of accelerating crystallization of insulin during scaling up, in the crystallization solution obtained from reverse phase chromatography during polishing stage, by application of reduced pressure after pH adjustment near isoelectric point.
Yet another object of the present invention is to develop a method to prepare pharmacopoeial grade insulin crystals by drying of the crystallized insulin under vacuum of 5 mbar to 50 mbar at 25°C to 45°C
Yet another object of the invention is a pharmaceutical composition comprising insulin crystals obtained by the said method.
Definition of Terms
The term "crystallization" as used herein refers to the formation of insulin crystals.

The term "insulin" refers to native insulin, animal origin insulin or recombinant human insulin, wherein recombinant human insulin is prepared by recombinant DNA technology.
The term "reverse phase chromatography" is the process of final purification of insulin 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 using "reverse phase chromatography".
The term "fractions" refers to the portions of elute collected from "reverse phase chromatography". Fractions contain pure insulin.
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 protein is zero wherein protein 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.
Detailed description of the Invention:
The first embodiment of the present invention relates to the method of crystallizing insulin in the fractions collected from the reverse phase chromatography during polishing stage. Crystallization of insulin is carried out without lyophilization and drying of the fractions collected from the reverse phase chromatography.
According to this embodiment, reverse phase chromatography elute fractions containing pure insulin are collected and are pooled together to obtain the "crystallization solution". Further the method of crystallization of insulin comprises the steps of:

a. optionally diluting crystallization solution to obtain crystallization solution
containing lower concentration of insulin;
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 metal ion to the acidified crystallization solution obtained in step 'b' to
prepare metal ion containing solution;
d. adding second organic solvent to metal ion containing solution obtained in step
'c' to prepare diluted solution;
e. adding inorganic base to diluted solution obtained in step 'd' and adjusting the
pH to 5 - 7 to prepare pH adjusted solution;
f. maintaining, while stirring, the pH adjusted solution obtained in step 'e' initially
at 20°C to 30°C for 6 hrs to 10 hrs and then at 2°C to 8°C for 48 hrs to 96 hrs for
complete crystallization.
"Crystallization solution" is referred as solution hereafter.
According to this aspect to the invention in step ca' 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 insulin as desired. The solution is then treated sequentially, with organic acid in step cb\ metal ion in step cc' and second organic solvent in step cd' and then it is subjected to crystallization near isoelectric point as in step 'e' and T.
Accordingly crystallization of insulin from the solution is initiated in step 'b' 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 citric acid. The 2M stock solution of Citric is added to the crystallization solution to obtain acidified crystallization solution in which final concentration of citric acid is 0.025 M to 0.1 M, preferably 0.052 M. Further according to this embodiment the source of metal ions

used for crystallization of insulin from the acidified crystallization solution obtained in step 6b' 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. The acidified crystallization solution is thus converted to metal ion containing solution in step ccAccording to step 'd' of this aspect of invention, second organic solvent is further added to the metal ion containing solution obtained in step V to prepare diluted solution. The diluted solution contains protein with decreased solubility and it stabilizes the crystal formation. The second organic solvent is selected from acetone, ethanol or methanol; preferably acetone.
In the method of crystallization of insulin, pH adjusted solution is prepared in step 'e\ from the diluted solution obtained in step cd' by adjusting the 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.
According to step T of this aspect of invention, the temperature of the pH adjusted solution obtained in step 'e' is maintained at 20°C to 25°C for 6 - 10 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 48 hrs to 96 hrs preferably 72 hrs to complete crystallization of insulin.
In second embodiment of the present invention insulin is crystallized in the crystallization solution obtained from reverse phase chromatography during polishing stage, wherein the crystallization solution comprises high quantities of organic solvent and wherein the concentration of insulin in the crystallization solution is in the range of 1.0 mg/mL to 10 mg/mL.

Further according to this embodiment insulin is crystallized by the method comprising the steps of:
a. optionally diluting crystallization solution to obtain crystallization solution
containing lower concentration of insulin;
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 metal ion to the acidified crystallization solution obtained in step 6b' to
prepare metal ion containing solution;
d. adding second organic solvent to metal ion containing solution obtained in step
'c' to prepare diluted solution;
e. adding inorganic base to diluted solution obtained in step 'd' and adjusting the
pH to 5 - 7 to prepare pH adjusted solution;
f. maintaining, while stirring, the pH adjusted solution obtained in step 'e' initially
at 20°C to 30°C for 6 hrs to 10 hrs and then at 2°C to 8°C for 48 hrs to 96 hrs for
complete crystallization.
According to this embodiment the crystallization solution obtained from reverse phase chromatography during polishing stage contains 25% to 35% of organic solvent. Crystallization is either carried out in the presence of 25% to 35% of organic solvent or it is optionally diluted as in step 'a' to obtain solution containing lower concentration of insulin. However direct crystallization without dilution of the solution, delays process of crystallization.
According to this embodiment, organic solvent is a non-aqueous solvent used in the reverse phase chromatography during polishing stage of insulin, which is selected from acetonitrile, methanol, ethanol or Isopropyl alcohol; preferably acetonitrile.
Further according to the step 'a' of this embodiment, the solution is diluted either using the mobile phase used in reverse phase chromatography or using acidified

water for injection, wherein acidified water comprises 0.4% acetic acid in water. After dilution the solution contains preferably 10% to 25%; more preferably 10% to 20% of organic solvent.
According to this embodiment, the solution in step 'a' is treated sequentially, with organic acid in step cb', metal ion in step 6c' and second organic solvent in step 'd' and then it is subjected to crystallization near isoelectric point as in step 'e' and T.
Accordingly crystallization of insulin from the solution is initiated in step 'b' 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 citric acid. The 2M stock solution of Citric is added to the crystallization solution to obtain acidified crystallization solution in which final concentration of citric acid is 0.025 M to 0.1 M, preferably 0.052 M. Further according to this embodiment the source of metal ions used for crystallization of insulin from the acidified crystallization solution obtained in step 'b' 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. The acidified crystallization solution is thus converted to metal ion containing solution in step 'c'
According to step 'd' of this aspect of invention, second organic solvent is further added to the metal ion containing solution obtained in step 'c' to prepare diluted solution. The diluted solution contains protein with decreased solubility and it stabilizes the crystal formation. The second organic solvent is selected from acetone, ethanol or methanol; preferably acetone.
In the method of crystallization of insulin, pH adjusted solution is prepared in step V, from the diluted solution obtained in step 'd' by adjusting the 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.
According to step 'f' of this aspect of invention, the temperature of the pH adjusted solution obtained in step 'e' is maintained at 20°C to 25°C for 6 - 10 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 48 hrs to 96 hrs preferably 72 hrs to complete crystallization of insulin.
The third embodiment of the present invention is a method of acceleration of crystallization of insulin 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.
According to this embodiment of the present invention, the method of crystallization of insulin described herein is carried out 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 insulin;
b. lowering the pH to less than or equal to 3 by acidifying the crystallization
solution in step a using organic acid to obtain acidified crystallization
solution;
c. adding metal ion to the acidified crystallization solution obtained in step b,
to prepare metal ion containing solution;
d. adding second organic solvent to metal ion containing solution obtained in
step c, to prepare diluted solution;
e. adding inorganic base to diluted solution obtained in step d and adjusting
the pH to 5 - 7 to prepare pH adjusted solution;
f. maintaining under reduced pressure, while stirring, the pH adjusted

solution obtained in step e, initially at 20°C to 30°C for 6 hrs to 10 hrs and then at 2°C to 8°C for 48 hrs to 96 hrs for complete crystallization.
According to this aspect to 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 insulin as desired. The solution is then treated sequentially, with organic acid in step 'b' metal ion in step 'c' and second organic solvent in step 'd' and then it is subjected to crystallization near isoelectric point as in step 'e' and 'f'.
Accordingly crystallization of insulin from the solution is initiated in step 'b' 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 citric acid. The 2M stock solution of Citric is added to the crystallization solution to obtain acidified crystallization solution in which final concentration of citric acid is 0.025 M to 0.1 M, preferably 0.052 M. Further according to this embodiment the source of metal ions used for crystallization of insulin from the acidified crystallization solution obtained in step 'b' 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. The acidified crystallization solution is thus converted to metal ion containing solution in step 'c'
According to step 'd' of this aspect of invention, second organic solvent is further added to the metal ion containing solution obtained in step 'c' to prepare diluted solution. The diluted solution contains protein with decreased solubility and it stabilizes the crystal formation. The second organic solvent is selected from acetone, ethanol or methanol; preferably acetone.
In the method of crystallization of insulin, pH adjusted solution is prepared in step 'e', from the diluted solution obtained in step 'd' by adjusting the 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.
According to step 'f' of this aspect of invention, the temperature of the pH adjusted solution obtained in step 'e' is maintained at 20°C to 25°C for 6 - 10 hrs. The method of crystallization, in step 'e' is accelerated by applying a constant reduced pressure of 100 to 400 mbar for 4 hrs to 6 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 48 hrs to 96 hrs preferably 72 hrs to complete crystallization of insulin. The method is reproducible with uncompromising quality and yield of the product.
Recovery of crystals obtained by any of the methods described herein is done by conventional methods like filtration under vacuum or centrifugation and the crystals collected are washed with chilled water for injection (WFI) or purified water followed by absolute alcohol or acetone.
Yet another embodiment of the present invention is a method to prepare pharmacopoeial grade insulin crystals by drying of the crystallized insulin under vacuum of 5 mbar to 50 mbar at 25°C to 45°C.
According to this embodiment of the invention, insulin crystals obtained after filtration/centrifugation stage are dried at 5 mbar to 50 mbar of vacuum at 35-45°C for 16-24 hrs to give a stable solid form of insulin suitable for holding and dispensing to fill/finish operations.
Both the parameters viz temperature and pressure were found to be critical to obtain pharmacopoeial grade insulin crystals. Higher temperature higher vacuum could destroy the crystals, whereas lower temperature and lower pressure yield insulin with residual solvents above pharmacopoeial limit. When the crystals were dried at 20 mbar pressure and at 32°C temperature, insulin 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 when dried under 5 mbar to 50 mbar of vacuum at 35-45°C preferably 38°C for 16-24 hrs could yield pharmacopoeial grade insulin having 60.5 ppm of acetone and acetonitrile below quantification limit.
Yet another embodiment of the invention is a pharmaceutical composition comprising insulin crystals prepared by the method described herein.
According to this embodiment, the pharmaceutical composition comprises crystalline insulin obtained by the method described herein and pharmaceutically acceptable excepients.
Further according to this embodiment the pharmaceutical composition is administered orally, 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.
Conventionally crystallization of insulin is carried out after lyophilization, evaporation, repeated concentration and drying of the fractions containing pure insulin. 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 is separated from solvent containing fractions and a solution of insulin 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, 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 is direct crystallization of insulin in the fractions collected from reverse phase chromatography, in presence of high amount of organic solvent. There is no prior art teaching wherein the crystallization of insulin is reported in a solvent system at such a low concentration of insulin.
Recombinant human insulin used for crystallization in this invention was prepared by expressing the precursor molecule preproinsulin in E.coli. The proinsulin was prepared from the said preproinsulin by solubilization of inclusion bodies followed by clarification, concentration and chemical digestion. Further Proinsulin 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 cleaved reaction mixture was purified by ion exchange chromatography to yield purified cleaved protein. The purified cleaved protein was further subjected to Carboxypeptidase B digestion to remove the C-Peptide to form insulin molecule. The insulin 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 with desired purity.
The following examples are provided merely to further illustrate the preparation of insulin crystals and they do not limit the invention in any manner.
Example 1: Crystallization in more than 20% acetonitrile at protein concentration of 2.4 mg/mL of insulin
1.68 gram polished pooled sample at 6.41 mg/mL was diluted with 22.5% of acetonitrile containing 0.4% acetic acid solution to bring down the concentration to 2.4 mg/ml. 18.2 mL of 2M Citric acid was added to lower the pH to 3 and incubated for 10 min. 2.01 mL of 20% w/v of zinc chloride solution was added followed by

addition of 70 mL of acetone. pH of the reaction mixture was adjusted to 6.17 by ammonia solution and kept under slow stirring .Crystals were observed after 5 hrs of agitation at 25°C. After 7 hrs from pH adjustment crystallization solution was shifted to 2-8°C and maintained at this temperature for 96 hrs. Crystals were recovered by membrane filtration under vacuum and washed with 350 mL of chilled WFI, followed by 135 mL of absolute alcohol. The crystals were collected in a Petri plate and dried at 38°C for 16 hrs under vacuum. 1.8 gms of crystals were obtained after drying and were analyzed as per 6 edition of Indian pharmacopoeia. The powder showed 99.04% Insulin purity, 0.52% other impurities, 0.44 % A-21 Des-amido impurity and potency as 29.31 IU/mg.
Example 2: Crystallization at around 25% acetonitrile
5.94 gms of polished pooled sample at 7.43 mg/mL was taken. 20.8 mL of 2M Citric acid was added to lower the pH of the pooled sample to 3 and incubated it for 10 min. 4.75 mL of 20% w/v of zinc chloride solution was added followed by addition of 80 mL of acetone. pH of the reaction mixture was adjusted to 6.17 by dilute ammonia solution and kept under slow stirring. Crystals were observed after 3 hrs of agitation at 25°C. After 5 hrs of pH adjustment crystallization solution was shifted to 2-8°C for and maintained at this temperature for 96 hrs. Crystals were recovered by membrane filtration under vacuum and washed with 800 mL of chilled WFI, followed by 160 mL of acetone. The crystals were collected in a Petri plate and dried at 38°C for 16 hrs under vacuum. 6.1 gms of crystals were obtained after drying and was analyzed as per 6l edition of Indian pharmacopoeia. The powder showed 98.3% Insulin purity, 1.4% other impurities, 0.3% A-21 Des-amido impurity and potency as 27.7 IU/mg.
Example 3: Crystallization at 1.0 mg/mL of insulin concentration in 10% acetonitrile: This experiment was carried out to test if the crystallization takes place at this concentration.

50 mg of polished pooled sample at 3.78 mg/mL was diluted to 1.5 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 1.0 mg/ml 1.3 mL of 2M Citric acid was added to lower the pH to 3 and incubated for 10 min. 40 microliter of 20% w/v of zinc chloride solution was added followed by addition of 5 mL of acetone. pH of the reaction mixture was adjusted to 6.18 by dilute ammonia solution and kept under slow stirring. Well defined crystals were observed after 9 hour.
Example 4: Crystallization in more than 20% acetonitrile at 7.5 mg/mL protein concentration
1.95 gms of polished pooled protein sample at 8.72 mg/mL was diluted with 22.5% of acetonitrile containing 0.4% acetic acid solution to bring down the concentration to 7.5 mg/mL. 6.89 mL of 2M Citric acid was added to lower the pH to 3 and incubated for 10 min. 1.6 mL of 20% w/v of zinc chloride solution was added followed by addition of 26 mL of acetone. pH of the reaction mixture was adjusted to 6.18 by ammonia solution and kept under slow stirring. Crystals were observed after 3 hrs of agitation at 25°C. After 4 hrs crystallization solution was shifted to 2-8°C and maintained at this temperature for 96 hrs. Crystals were recovered by membrane filtration under vacuum and washed with 220 mL of chilled WFI, followed by 100 mL of absolute alcohol. The crystals were collected in a Petri plate and dried at 38°C for 16 hr under vacuum. 2.11 gm of crystals were obtained after drying and were analyzed as per 6 edition of Indian pharmacopoeia. The powder showed 98.91% Insulin purity, 0.79% related impurities, 0.39% A-21 Des-amido impurity and potency as 28.41 IU/mg
Example 5: Illustrates the crystallization on pilot scale
35.7 gms of polished pooled sample at 6.6 gm/L was taken and diluted with 1.73L of WFI containing 0.4% acetic acid to a final concentration of 5 g/L. 186 mL of 2M

Citric acid was added to lower the pH to 3 and incubated for 10 min. 28.6 mL of 20% w/v of zinc chloride solution was added followed by addition of 715 mL of acetone. pH of the reaction mixture was adjusted to 6.18 by dilute ammonia solution and kept under slow stirring with a constant reduced pressure of 200 mbar for 6.0 hrs at 25°C and then shifted to 2-8°C for 72 hrs. Crystals were recovered by membrane filtration under vacuum and washed with 7.0L of chilled WFI, followed by 1.0L of acetone. The crystals were collected in a Petri plate and dried at 38°C for 24 hr under vacuum of 10 mbar. 35.7 gm of crystals were obtained after drying and were analyzed as per 6 edition of Indian pharmacopoeia. The powder showed 99.26% Insulin purity, 0.34% other impurities, 0.4% A-21 Des-amido impurity and potency as 29.3 IU/mg and the residual solvent for acetone and acetonitrile were 1517 ppm and 62.7 ppm respectively.
Example 6: Drying of Crystalline Insulin
28 gms of polished pooled sample at 8.77 g/L was taken and diluted with 2.41L of WFI containing 0.4% acetic acid to a final concentration of 5 g/L. 146 mL of 2M Citric acid was added to lower the pH to 3 and incubated for 10 min. 22.4 mL of 20% w/v of zinc chloride solution was added followed by addition of 560 mL of acetone. pH of the reaction mixture was adjusted to 6.19 by dilute ammonia solution and kept under slow stirring with a constant reduced pressure of 200 mbar for 6 hrs at 25°C and then shifted to 2-8°C for 72 hrs. Crystals were recovered by membrane filtration under vacuum and washed with 5.6L of chilled WFI, followed by 0.56L of acetone. The crystals were collected in a Petri plate and dried at 38°C for 17 hr under vacuum of 5 mbar. 27.6 gms of crystals were obtained after drying and were analyzed as per 6th edition of Indian pharmacopoeia. The powder showed 99.33% Insulin purity, 0.33% other impurities, 0.34% A-21 Des-amido impurity and potency as 28.73 IU/mg. Residual solvent for acetone and acetonitrile were 60.5 ppm and below quantification limit respectively and the moisture content (estimated as Loss on

drying) in the final drug substance was 6.25%, which is within pharmacopoeia! limit (Not more then 10%)

We Claim
1. A method of crystallizing insulin in the crystallization solution obtained from reverse phase chromatography during polishing stage, wherein the insulin is not isolated from the crystallization solution obtained from reverse phase chromatography during polishing stage.
2. The method as claimed in claim 1, comprises the steps of:
a. optionally diluting crystallization solution to obtain crystallization
solution containing lower concentration of insulin;
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 metal ion to the acidified crystallization solution obtained in step b,
to prepare metal ion containing solution;
d. adding second organic solvent to metal ion containing solution obtained in
step c, to prepare diluted solution;
e. adding inorganic base to diluted solution obtained in step d and adjusting
the pH to 5 - 7 to prepare pH adjusted solution;
f. maintaining, while stirring, the pH adjusted solution obtained in step e,
initially at 20 °C to 30°C for 6 hrs to 10 hrs and then at 2°C to 8°C for 48
hrs to 96 hrs for complete crystallization.
3. The method as claimed in claim 2a, wherein the crystallization solution is diluted using either mobile phase of reverse phase chromatography or using 0.4% acetic acid in water for injection.
4. The method as claimed in claim 2b, wherein organic acid is selected from citric acid, acetic acid, lactic acid, oxalic acid; preferably citric acid with 2M concentration.

5. The method as claimed in claim 2c, wherein the metal ion is selected from Zinc ions, Cadmium ions or Cobalt; in the form of their halides, acetates or sulphate; preferably Zinc ion as Zinc Chloride.
6. The method as claimed in claim 5, wherein the concentration of Zinc Chloride is in the range of 8% to 24% w/w; preferably 16% w/w.
7. The method as claimed in claim 2d, wherein the second organic solvent is selected from acetone, ethanol and methanol; preferably acetone.
8. The method as claimed in claim 2e, wherein the inorganic base is selected from Sodium Hydroxide, Potassium Hydroxide Calcium Hydroxide or Ammonia solution; more preferably ammonia solution; and wherein the preferred pH of the pH adjusted solution is 5.8 to 6.4.
9. A method of crystallizing insulin in the crystallization solution obtained from reverse phase chromatography during polishing stage, wherein the crystallization solution comprises from 10% to 35% of organic solvent; and wherein the concentration of insulin in the crystallization solution is in the range of 1.0 mg/mL to 10 mg/mL.
10. The method as claimed in claim 9, wherein the crystallization solution preferably comprises 10% to 25% of organic solvent; more preferably 10% to 20% of organic solvent, and the preferred concentration of insulin in the crystallization solution is in the range of 1.0 mg/mL to 7.5 mg/mL.
11. The method as claimed in claim 9, comprises the steps of:
a. optionally diluting crystallization solution to obtain crystallization
solution containing lower concentration of insulin;
b. lowering the pH to less than or equal to 3 by acidifying the crystallization
solution in step a using organic acid to obtain acidified crystallization
solution;

c. adding metal ion to the acidified crystallization solution obtained in step b?
to prepare metal ion containing solution;
d. adding second organic solvent to metal ion containing solution obtained in
step c, to prepare diluted solution;
e. adding inorganic base to diluted solution obtained in step d and adjusting
the pH to 5 - 7 to prepare pH adjusted solution;
f. maintaining, while stirring, the pH adjusted solution obtained in step e,
initially at 20 °C to 30°C for 6 hrs to 10 hrs and then at 2°C to 8°C for 48
hrs to 96 hrs for complete crystallization.
12. The method as claimed in claim 11a, wherein the crystallization solution is diluted using either mobile phase of reverse phase chromatography or using 0.4% acetic acid in water of injection.
13. The method as claimed in claim 1 lb, wherein organic acid is selected from citric acid, acetic acid, lactic acid, oxalic acid; preferably citric acid with 2M concentration.
14. The method as claimed in claim l1c, wherein the metal ion is selected from Zinc ions, Cadmium ions or Cobalt ions; in the form of their halides, acetates or sulphate; preferably Zinc ions as Zinc Chloride.
15. The method as claimed in claim 14, wherein the concentration of Zinc Chloride is in the range of 8% to 24% w/w; preferably 16% w/w.
16. The method as claimed in claim l1d, wherein the second organic solvent is selected from acetone, ethanol and methanol; preferably acetone.
17. The method as claimed in claim l1e, wherein the inorganic base is selected from Sodium Hydroxide, Potassium Hydroxide Calcium Hydroxide or Ammonia solution; more preferably ammonia solution; and wherein the preferred pH of the pH adjusted solution is 5.8 to 6.4.

18. A method for acceleration of crystallization of insulin 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.
19. The method as claimed in claim 18, comprises the steps of:
g. optionally diluting crystallization solution to obtain crystallization solution containing lower concentration of insulin;
h. lowering the pH to less than or equal to 3 by acidifying the crystallization solution in step a using organic acid to obtain acidified crystallization solution;
i. adding metal ion to the acidified crystallization solution obtained in step b, to prepare metaltion containing solution;
j. adding second organic solvent to metal ion containing solution obtained in step c, to prepare diluted solution;
k. adding inorganic base to diluted solution obtained in step d and adjusting the pH to 5 - 7 to prepare pH adjusted solution;
1. maintaining under reduced pressure, while stirring, the pH adjusted solution obtained in step e, initially at 20 °C to 30°C for 6 hrs to 10 hrs and then at 2°C to 8°C for 48 hrs to 96 hrs for complete crystallization.
20. The method as claimed in claim 19a, wherein the crystallization solution is diluted using either mobile phase of reverse phase chromatography or using 0.4% acetic acid in water of injection.
21. The method as claimed in claim 19b, wherein organic acid is selected from citric acid, acetic acid, lactic acid, oxalic acid; preferably citric acid with 2M concentration.
22. The method as claimed in claim 19c, wherein the metal ion is selected from Zinc

ions, Cadmium ions or Cobalt ions; in the form of their halides, acetates or sulphate; preferably Zinc ions as Zinc Chloride.
23. The method as claimed in claim 22, wherein the concentration of Zinc Chloride is in the range of 8% to 24% w/w; preferably 16% w/w.
24. The method as claimed in claim 19d, wherein the second organic solvent is selected from acetone, ethanol and methanol; preferably acetone.
25. The method as claimed in claim 19e, wherein the inorganic base is selected from Sodium Hydroxide, Potassium Hydroxide Calcium Hydroxide or Ammonia solution; more preferably ammonia solution; and wherein the preferred pH of the pH adjusted solution is 5.8 to 6.4.
26. The method as claimed in claim 18, wherein the pressure is preferably reduced to 200 mbar for 4 hrs to 6 hrs.
27. The method as claimed in claim 19f, wherein the pressure is preferably reduced to 200 mbar for 4 hrs to 6 hrs.
28. A method of preparation of pharmacopoeial grade insulin crystals, wherein the method comprises drying insulin crystals for 16 to 24 hrs under vacuum of 5 mbar to 50 mbar at 25° C to 45°C.
29. The method as claimed in claim 28, wherein drying is preferably carried out under vacuum of 5 mbar to 50 mbar at 35°C to 45°C more preferably 38°C.
30. The pharmaceutical composition comprising crystallized insulin obtained by the method as claimed in any preceding claims.