Patent Application
20220064213
The present invention relates to a method for peptide crystallization, particularly preparing recombinant human insulin crystal.
Diabetes is a common endocrine and metabolic disorder. It's a long-term condition that causes high blood sugar levels. In type 1 Diabetes, the body does not produce Insulin, also referred to as Insulin-dependent diabetes, juvenile diabetes, or early-onset diabetes. In type 2 Diabetes, the body does not produce enough insulin for proper function, or the cells in the body do not react to Insulin (Insulin resistance). Presently patients with type 1 diabetes are treated with regular insulin injections along with a special diet and exercise. Patients with Type 2 diabetes are treated with tablets, exercise and a special diet, but sometimes insulin injections are also required.
‘Insulin therapy’ has always been considered as an important means for treating diabetes and controlling blood sugar level. Purification of recombinant Human Insulin achieved by multiple downstream unit operations that involve a combination of crystallization, enzyme catalysis and chromatography. The requirement of the highest purity of Human Insulin is to ensure the patients do not develop immunogenic or toxic responses to the drug product.
Recombinant Human Insulin crystallization occurs in two phases. The first phase is nucleation, the appearance of a crystalline phase from either a super cooled liquid or a supersaturated solvent. The second phase is crystal growth, which is the increase in the size of particles and leads to a crystalline state. The crystal form of recombinant Human Insulin is a better form, since it has a uniform and steady solid molecular form and small sediment volume, and is easy to separate from the supernatant, the time for centrifugation and freeze-drying is short, and the production efficiency is relatively high. It is thus desirable to prepare recombinant Human Insulin crystals and then apply the crystals to Insulin pharmaceutical preparations.
Commercial Insulin manufacturing processes typically include a crystallization step to convert soluble purified Insulin into solid form, providing increased stability for bulk storage prior to formulation and filling. Classical Insulin crystallization process as disclosed in U.S. Pat. No. 2,910,014 includes preparation of an acidic solution containing organic acid (acetic or citric), approximately 2 g/L Insulin, and zinc and adjustment of the solution pH to near the isoelectric point of insulin (pH 5.5-6.0), which initiated crystal formation.
It is well known in the art that Insulin may be crystallized in the presence of zinc ions, resulting in a crystalline preparation with significant benefits over amorphous, un-crystallized Insulin with regard to stability, storage, formulation, and/or administration. In the presence of zinc, human insulin self-assembles into stable hexameric structures. Zinc content plays an important role in chemical and physical stability of pharmaceutical insulin formulations.
The use of ZnCl2 for crystallization of recombinant Human Insulin (rHI) is an established and well published technique, however, a similar knowledge is not available for crystallizing HI at a preparative scale. Traditional recombinant Human Insulin downstream purification process involves three crystallization steps termed as crystallization-1, crystallization-2 and crystallization-3, respectively as per the order of the operation. Controlling the level of aggregates, residual zinc and other related impurities during drug preparation is a critical quality requirement to make the final drug product complying with the specifications of the innovator. In the recent manufacturing batches of the biosimilar Insulin process, it was noted that level of high molecular weight protein (HMWP) and other related impurities were on the higher level. Crystallization-3 is a potential step, which is a final step of crystallization, where the chances of formation of aggregates (HMWP) and other related impurities are imminent. Improper settling during the neat settling step was observed during this final crystallization stage(s). This suboptimal performance leads to the lower decantation percentage at neat and wash-1 stage resulting in suboptimal freeze-drying performance.
Therefore, there is a need to control the level of these impurities at the appropriate downstream step.
An object of the present invention is to overcome the various key process challenges observed during the final crystallization & freeze drying stages of recombinant Human Insulin preparation and accordingly modify the process.
Another object of the present invention of preparative peptide crystallization is to obtain consistent crystal geometry and size of recombinant Human Insulin at preparative scale.
In one aspect the present invention provides a method for preparing recombinant Human Insulin crystal comprising the steps of:
In another aspect the present invention provides a method comprising the steps of:
Unless otherwise defined herein; the scientific and technical terms used in connection with the present invention shall have the meanings that are, commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art. The nomenclatures used in connection with, and techniques described herein are those commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art.
The term ‘ambient temperature’ refers to the air temperature of an environment or object or surrounding an equipment. Room Temperature (RT) is generally defined as the ambient air temperature. In present invention, ambient temperatures or room temperature can range between 20 and 26° C.
The term ‘crystallization’ refers to the solid-liquid separation and purification technique in which mass transfer occurs from the liquid solution to a pure solid crystalline phase.
The term ‘Insulin’ refers to a hormone secreted by the islets of Langerhans in the pancreas; regulates storage of glycogen in the liver and accelerates oxidation of sugar in cells.
The term ‘recombinant Human Insulin’ and ‘rHI’ refer to a form of insulin made from recombinant DNA that is identical to human insulin.
The present inventors studied the process deviations for multiple batches at final crystallization and freeze-drying step and found that all deviations were due to the poor neat settling during the final crystallization process (also corroborated by the smaller crystal size i.e. <1 μm). After a thorough root cause analysis (elaborated through the examples below), following factors have been identified to be affecting the crystal size and subsequent settling—
Further, freeze drying failure (improper product drying) was primarily due to the cascading effect of the suboptimal settling in preceding crystallization step. This resulted in higher slurry volume post wash decantation that was almost 12% higher than the expected volume. This led to the loading of each freeze-drying tray with higher bed height and lower slurry percentage (5%). It was found that lower ambient temperature resulted in poor settling while higher hold duration would lead to increase in aggregates (HMWP). It was further observed that at lower slurry percentage higher bed height is detrimental for efficient product drying.
In order to mitigate the problems of the old process, the present invention provides an improved method for preparing recombinant Human Insulin crystal comprising the steps of crystallizing the recombinant Human Insulin in a crystallization solution containing recombinant Human Insulin, an organic solvent, a zinc compound, a salt, such that, the mixture of zinc compound and the salt are added together in crystallization solution followed by a pH adjustment to 4.8 to 5.2, preferably 5.0.
The present invention, particularly provides an improved crystallization-3 process with FFC targeting 21 million IPA content (ppm).
The concentration of the recombinant human insulin in the crystallization solution is 5.0±0.2 g/L.
In the present invention, the organic solvent is selected from acetonitrile, ethanol, n-propanol and isopropyl alcohol, preferably isopropyl alcohol. The concentration of isopropyl alcohol is from 19 to 25 million ppm, preferably 21 million ppm.
The zinc compound is selected from zinc chloride, zinc oxide, zinc acetate, zinc bromide and zinc sulfate. The salt is selected from sodium chloride, sodium acetate and sodium citrate. Preferably, the solution contains 4% zinc chloride at a concentration of 0.3-0.5 ml per gram of recombinant Human Insulin and 0.5M sodium chloride, which is added as a solution to the crystallization solution.
The pH of the crystallization solution was adjusted in the range of 4.8 to 5.2 using 3M acetic acid within 5 minutes after addition of mixture of zinc chloride and sodium chloride mixture in the crystallization solution.
In a preferred embodiment, the crystallization solution is held at the ambient temperature of 24±3° C. for 2.5 to 4 hours for neat settling, upon which, the chilling of neat settling is achieved at 2-8° C. for 10-12 hours. Further, the slurry is held at 2-8° C. for another 16 hours. The slurry obtained therein is freeze dried.
Elution pool of HPLC was diluted by WFI and IPA followed by addition of solution of ZnCl2+NaCl mixture. The adjustment of 5.0 pH was achieved within 5 minutes of such addition. It was surprisingly found that the faster rate of pH adjustment and the maintenance of ambient/room temperature is vital for protein crystallization, settling and consistent crystal size. The steps of neat settling and complete wash are performed at cold temperature (5±3° C.), which adds to the process robustness and better control of critical quality attributes at final drug substance stage. The 1.1 cm bed height of loading tray used during freeze-drying step for efficient product drying.
In a particular embodiment, the present invention provides a method comprising steps of:
The present method yields recombinant Human Insulin having a consistent crystal size of about 15 μm-30 μm. It also reduces the time required for sedimentation at manufacturing scale to about 12 hours compared to traditional process wherein the sedimentation step requires 24-60 hours whereas the present invention achieves the same in 12 hours.
Table 1 elaborates the material and the grade of material used for the altering of crystallization-3 process for crystallizing recombinant human insulin.
Table 2 elaborates the reagents and method of its preparation used for the present invention of altering of crystallization-3 process for crystallizing recombinant human insulin.
Table 3 elaborates the analytical method(s) used for the altering of crystallization-3 process for crystallizing recombinant human insulin. The table also elaborates the stage of process during which the particular method been used.
Table 4 elaborates the name and model of the equipment used for the altering of crystallization-3 process for crystallizing recombinant human insulin.
Table 5 elaborates the preparative and analytical columns used for the altering of crystallization-3 process for crystallizing recombinant human insulin.
Table 6 elaborates revised crystallization-3 process parameters and ranges followed in present invention related to the altering of crystallization-3 process for crystallizing recombinant human insulin.
Parts per million (ppm) calculations used for measuring small concentrations in a solution. In present invention, it was required to prepare an accurate of amount blank buffer (L) for dilution of RP-HPLC3 Elution Pool (EP) (5.0±0.2 g/L) based on the formula elaborated in table 7 below.
The present invention is further elaborated with the help of following examples. However, these examples should not be construed to limit the scope of present invention.
Haziness was observed in the FFC (feed for crystallization) upon NaCl addition during the satellite trials conducted at the laboratory. NaCl used to hasten the rate of settling during the crystallization process. Thus, based on this attributed purpose of NaCl, experiments were performed to understand whether the change in chronology of NaCl addition would mitigate the haziness observed in the FFC of the batch.
The reagent addition chronology was studied in two strategies viz.
Strategy 1: Addition of 0.5M NaCl post pH adjustment of the crystallization mixture.
Strategy 2: Addition of 4% ZnCl2+0.5M NaCl mixture to the FFC at 5.0 g/L.
The Strategy 1 is Addition of 0.5M NaCl post pH adjustment of the crystallization mixture elaborated as follows in table 8 and 9.
Table 8 elaborates the details of the reagent addition performed in strategy 1. In strategy 1, the RP-HPLC 3 elution pool (EP) of 8.70 g/L concentration was diluted to 5.0 g/L with WFI. This dilution was followed by addition of ZnCl2, which was immediately followed by pH adjustment to 5.0±0.1. NaCl was added to the mixture and it was held at 24±3° C. until neat settling. Table 9 elaborates the details of the observations on reagent addition of strategy 1.
From the trial experiments T5, T6, T20 & T21 (shown in
The Strategy 2 is Addition of 4% ZnCl2+0.5M NaCl mixture to the FFC at 5.0 g/L elaborated as follows in table 10 and 11.
Strategy 2 was explored due to the inconsistent results of the strategy 1. Table 10 elaborates the details of the reagent addition performed for strategy 2. In this strategy, experiments were performed by mixing the required amount of 4% ZnCl2 and 0.5M NaCl mixture and in turn added to the prepared FFC at 5.0 g/L. Following which crystallization-3 was performed by adjusting the pH with 3.0M Acetic acid to 5.0±0.1. Table 11 elaborates the details of the observations on reagent addition of strategy 1.
From the trial strategy 2 experiments (shown in
Based on the observations in reagent addition chronology experiments and the observed variation in IPA content (ppm) in FFC; trials were performed by normalizing the IPA content (ppm) in FFC to hasten the settling rate during neat settling.
Table 12 elaborates the RP-HPLC3 EP dilution by targeting IPA content (ppm) in FFC whereas table 13 and
The IPA content (ppm) in FFC is vital for achieving desired settling and crystal size. However, the repetition of IPA content target experiments did not result in the similar crystal size. This observation led to study the rate of pH adjustment as one of the key factor that might influence crystal size.
Based on the varying crystal sizes (μm) observed despite appropriate addition of reagents (NaCl+ZnCl2) (discussed in example 1) and targeting IPA (ppm) in FFC (discussed in example 2), experiments elaborated in table 14 were performed to understand the impact of rate of pH adjustment.
The IPA content targeted to 21 million in FFC. The addition of 3M acetic acid for attaining the crystallization pH to 5.0±0.1 has to be 0.1 vvm irrespective of the scale of crystallization (refer Table 15).
To challenge the RP-HPLC3 product binding capacity as well as to understand the impact of RP-HPLC 3 product dynamic binding capacity (DBC) at final crystallization stage, RP-HPLC3 trials were performed at two different DBCs, one at 23 g/L and another one at 50 g/L to accommodate and propose the wider range of DBC at manufacturing scale unlike the current control limit for RP-HPLC 3 DBC (25 to 40.0 g/L).
RP-HPLC 2 load was procured from the manufacturing facility and RP-HPLC 2 and RP-HPLC 3 steps were performed at pilot lab as per insulin biosimilar process. All the individual fractions (after RPHPLC3) were adjusted to 7.35±0.1 after the elution. After pooling the fractions, pH of RPHPLC3 bulk EP was checked and adjusted to 7.4±0.1. Individual fractions pH essentially were not less than 7.3, as it may trigger protein precipitation.
Table 16 elaborate the RP-HPLC3 Load purity profile wherein table 17 elaborate RP-HPLC3 process performance and quality attributes at different DBC.
Quality attributes of RP-HPLC3 EP, which was generated at different DBC were comparable with control specifications. The same RP-HPLC 3 EP was utilized for crystallization-3 experiments, found no impact on crystallization process.
RP-HPLC3 EP was used for scale up trial.
From the various earlier trials (referred in example 1-4), it was observed that the reagent addition chronology, IPA content (ppm), pH adjustment rate and ambient hold temperature are vital for crystallization-3 process.
Based on the understanding, gained from various experiments, scale up trials were designed while keeping following process parameters under check (Table 18). Table 19 elaborates the scale up performance with respect to process & quality wherein
Due to the poor settling at neat settling stage during the Crystallization 3 stage of Insulin biosimilar process, lower decantation was performed at each stage (neat and wash). As per the general trend (data obtained upon following old process mentioned in
To understand the impact of Slurry percentage and bed height on Freeze-drying efficiency, the following trial elaborated in table 20 was designed. Manufacturing slurry (re-dissolved slurry) was procured to understand the impact of bed height & slurry percentage on freeze drying efficiency (Table 21).
From the results, it was quite evident that, at lower slurry percentage (4-5%), increase in bed height can impact on the moisture content (LOD value). Higher the bed height (with lower slurry percentage) in freeze drying tray would result in inefficient drying and sublimation.
For a particular crystallization condition (as captured in the ‘condition’ section of each table), observations are captured pertaining to each unique experiment. Table 22 elaborates the summary of final drug substance from revised crystallization process whereas table 23 elaborates the list of critical process parameter and its impact observed during the trails conducted (referred in example 1-6).
Based on the above data, it's evident that performing crystallization-3 process with FFC targeting 21 million IPA content (ppm), followed by addition of ZnCl2+NaCl mixture, & comparatively faster rate of pH adjustment and ambient temperature hold are vital for protein crystallization, settling and consistent crystal size. Performing remaining neat settling and complete wash, both at cold temperature (5±3° C.) can certainly add up to the process robustness and better control of critical quality attributes at final drug substance stage.
The process thus reduced time for sedimentation at manufacturing scale. In traditional process, the sedimentation used to take up to 24-60 hours whereas the present invention achieves the same result in 12 hours. The results arc shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 201941001190 | Jan 2019 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/050135 | 1/9/2020 | WO | 00 |
