Freeze-dried composition of pegaspargase

Abstract

To provide a new and economical freeze-dried composition of pegaspargase having storage stability.SOLUTION: The compositions of the present invention are comprised of pegaspargase, a cryoprotectant, a bulking agent, a buffer, and may optionally contain other pharmaceutically acceptable excipients including, but not limited to, salts. The compositions of the present invention are stable for extended periods of time at significant temperature ranges without the presence of significant amounts of impurities.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceutical science, and in particular to a lyophilized composition of pegaspargase and a process for its preparation. [Background technology]

[0002] Protein drug delivery poses a significant challenge to the biopharmaceutical industry due to the inherent instability of proteins in vivo. While orally administered proteins are easily digested in the gastrointestinal tract, parenterally injected proteins are generally prone to renal clearance and proteolysis. Protein drugs also face various challenges, including low solubility, short circulatory half-life, immunogenicity, and aggregation. As a result, protein persistence in the body is compromised. Several approaches have been attempted to achieve protein persistence in the body, including altering the amino acid sequence to reduce immunogenicity, removing proteolytic sites, conjugating to serum proteins, fusion with antibodies, incorporating into liposomes for sustained release, and conjugating to natural or synthetic polymers.

[0003] Conjugation of therapeutic proteins with polymers such as polyethylene glycol (PEG) has a long history in the biopharmaceutical industry and is accepted as a safe method for modifying proteins to extend their circulating half-life or reduce immunogenicity; information on this subject is available in US 4,179,337. This conjugation process is called PEGylation. PEG is classified as a "Generally Recognized as Safe" (GRAS) compound by regulatory bodies such as the USFDA and WHO.

[0004] PEG is a linear or branched polymer that is water-soluble (solubility increases with increasing molecular weight), lipophilic, and nontoxic. The lipophilic nature of PEG makes it suitable for end-group functionalization for conjugation to therapeutic proteins. Each PEG molecule typically binds two to three water molecules per ethylene oxide unit. PEGylation masks the protein surface and increases the molecular size of the polypeptide, preventing access by antibodies and antigen-processing cells and reducing degradation by proteolytic enzymes, thereby extending the circulatory half-life. Furthermore, the increased size (due to an increase in hydrodynamic radius) prolongs its circulation time by reducing renal clearance.

[0005] Many types of cancer, such as acute lymphocytic leukemia (ALL), are unable to synthesize the amino acid L-asparagine de novo (due to a lack or insufficiency of asparagine synthetase, which catalyzes the enzymatic conversion of the amino acid L-aspartic acid to L-asparagine), and instead absorb it from the blood and use it for cell proliferation. L-asparaginase is an enzyme that catalyzes the hydrolysis of L-asparagine to L-aspartic acid, releasing ammonia. By reducing L-asparagine levels in the blood, L-asparaginase inhibits L-asparagine uptake by cancer / tumor cells, ultimately leading to cell death. L-asparaginase can be isolated from bacteria, yeast, fungi, actinomycetes, and plants. L-asparaginase is useful for the treatment of tumors and cancers that depend on L-asparagine for protein synthesis. It is particularly used in the treatment of leukemias, such as acute lymphocytic leukemia, and is usually used in combination with other antitumor or anticancer agents, although it can also be used alone in certain clinical settings.

[0006] However, L-asparaginase itself has drawbacks due to its nature as a protein, including rapid clearance, a short half-life, proteolytic degradation, and the potential for non-human immune responses in patients treated with the enzyme. These drawbacks limit its long-term or repeated administration. As mentioned previously, these issues can be overcome by PEGylation. L-asparaginase (derived from Escherichia coli) is modified by covalently attaching a 5 kDa monomethoxypolyethylene glycol (mPEG). This PEG-asparaginase has the advantage of being virtually non-antigenic and having a reduced clearance rate from the circulatory system.

[0007] Pegaspargase, a liquid formulation at a concentration of 750 IU / mL, was approved by the U.S. FDA in 1994 for the treatment of acute lymphoblastic leukemia in patients with hypersensitivity to L-asparaginase and marketed under the trade name Oncaspar®. It was subsequently approved in 2006 as a first-line treatment for acute lymphoblastic leukemia as part of a multidrug combination therapy. Oncaspar® is manufactured by pegylation of 5 kDa monomethoxypolyethylene glycol (mPEG) succinimidyl succinate PEG (also known as SS-PEG). PEGylated asparaginase is disclosed in U.S. Patent Nos. 5,122,614; 5,324,844; 5,612,460; U.S. Patent No. 20120100121A1; and CN105802946A.

[0008] Despite these advantages, liquid compositions of pegaspargase have been reported to have problems such as thermal stability, difficulty in maintaining cold chain distribution, and a short shelf life. PEGylated proteins, especially those linked with a succinic acid linker, tend to degrade in liquid compositions, and in aqueous compositions, the ester bond between PEG and the succinic acid linker is hydrolyzed to free PEG and succinated protein. Clinical use of such important pharmaceuticals requires compositions that can be stored for long periods and can withstand temperature changes during manufacturing and delivery to clinics.

[0009] In many cases, stability issues associated with proteins in liquid compositions can be overcome by preparing them as solid compositions. It is well known that removing water is effective because all major degradation reactions (deamidation, hydrolysis, proteolysis, etc.) occur in aqueous solutions. The most common method used for liquid-to-solid conversion is freeze-drying or lyophilization. Lyophilization is a process that stabilizes pegaspargase and helps overcome the challenges. More than half of commercialized biological therapeutics are provided as lyophilized compositions.

[0010] The lyophilization cycle consists of three main steps: freezing, primary drying, and secondary drying, with an optional annealing step between freezing and primary drying. The lyophilization process is not stress-free and does not necessarily guarantee an extended shelf life for biopharmaceuticals. The stresses associated with the lyophilization step can cause physical (denaturation, aggregation, precipitation, etc.) and chemical (oxidation, Maillard reaction, covalent aggregation, etc.) degradation of proteins. These degradation pathways, which ultimately lead to loss of biological activity, are not mutually exclusive; in many cases, one leads to another, and both degradation pathways are somewhat interrelated.

[0011] The design of the lyophilization cycle is determined by the protein concentration and the nature and amount of bulking agents, stabilizers, and other excipients in the composition. Important thermal parameters, such as the apparent glass transition temperature (Tg') and the crystallization temperature of bulking agents, are usually determined for the composition prior to process design, as they guide the setting of temperature and pressure parameters for each step, including ramp and hold times at each stage of the lyophilization cycle.

[0012] PEGylated proteins present other complex issues that must be addressed to determine the final lyophilization step, such as the state of the PEG (amorphous or crystalline), the amount of free water available for interaction, storage temperature, lyophilization parameters, and protein to PEG ratio, all of which affect the activity of the protein after lyophilization, so no universal solution exists for all PEGylated products.

[0013] Therefore, it is important to tailor a unique process and composition for each protein, as a process or composition that works for one protein may not be effective for another.

[0014] US 6,180,096 and US 7,632,491B2 disclose compositions of PEG-interferon 2b with a longer freeze-drying cycle and a higher water content. US 8,367,054 B2 discloses compositions of PEG-interferon 2b with a shorter freeze-drying cycle. These documents disclose the importance of the freeze-drying process and suggest that product quality varies depending on the freeze-drying cycle.

[0015] CN105796507A discloses a stable composition of pegaspargase containing sorbitol, a protective agent, a buffer, and a surfactant. However, this composition has problems with stability in liquid form and protection during freezing. The application fails to provide a stable freeze-dried composition.

[0016] WO2018017190 discloses a freeze-dried, storage-stable composition, which comprises a polyalkylene oxide-asparaginase comprising a polyalkylene oxide group covalently bound to L-asparaginase via a linker, a buffer, a salt, and a sugar.

[0017] However, the process disclosed in WO'190 is time-consuming (up to 5 days) and uneconomical, and furthermore, the large amount of excipients used can undesirably increase the cost of the excipients by as much as 50%, potentially increasing the cost of the final product.

[0018] Pegaspargase is classified as an orphan drug and is expensive, so producing a shelf-stable product requires lyophilization costs and the addition of additives, which increases the cost of the product.

[0019] Therefore, there is a need for optimal storage-stable lyophilized compositions of pegaspargase that maintain their physical properties and biological activity during storage, and a lyophilization process for such compositions.

[0020] Object of the invention The object of the present invention is to provide a freeze-dried composition with optimal storage stability comprising pegaspargase that exhibits physicochemical stability and biological activity during storage, and a freeze-drying process for such a composition. Summary of the Invention

[0021] The present invention provides a freeze-dried composition with optimal storage stability comprising pegaspargase that exhibits physicochemical stability and biological activity during storage, and a freeze-drying process for such a composition.

[0022] The compositions of the present invention are stable over a significant temperature range for extended periods of time without the presence of significant amounts of impurities / degradants. The present invention also relates to an economically viable and scalable freeze-drying process for producing shelf-stable compositions of pegaspargase. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows a solid material structure for a composition within the scope of the present invention. [Figure 2]Figure 2 shows analytical data demonstrating the integrity and purity of pegaspargase before and after freezing (Figure 2(B)), as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion high-performance liquid chromatography (SE-HPLC). [Figure 3] Figure 3 shows analytical data assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) comparing the stability of a prior art liquid composition of pegaspargase (Figure 3(A)) with a lyophilized composition of the present invention (Figure 3(B)). [Figure 4] Figure 4 shows analytical data evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with Coomassie staining (Figure 4(A)) and iodine staining (Figure 4(B)), demonstrating the presence of high molecular weight impurities in the prior art PEG asparaginase lyophilized compositions compared with the lyophilized compositions of the present invention. Figures 4(C) and 4(D) show Western blot analysis of the samples against anti-asparaginase and anti-PEG antibodies, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description of the Invention composition The present invention provides an optimal storage-stable lyophilized composition containing pegaspargase that exhibits physicochemical stability and biological activity during storage, and a lyophilization process for such a composition.

[0025] The lyophilized composition of the present invention contains pegaspargase as an active ingredient. The lyophilized composition of the present invention includes pegaspargase, a cryoprotectant, a bulking agent, a buffer, and optionally other pharmaceutically acceptable excipients, including, but not limited to, salts.

[0026] The lyophilized composition of the present invention comprises a pegylated asparaginase in which a polyalkylene oxide group is covalently attached to the asparaginase via a linker.

[0027] The compositions of the present invention focus on PEGylated asparaginase, also known as pegaspargase, which comprises monomethoxypolyethylene glycol (mPEG) of molecular weight preferably 4 to 6 kDa, more preferably 4.5 to 5.5 kDa, and most preferably 4.8 to 5.2 kDa, covalently attached to one or more primary amine groups (the ε-amino acid and terminal amine of the lysine side chain) of L-asparaginase via an amide bond with a succinic acid linker.

[0028] L-asparaginase may be naturally obtained from Escherichia coli or other bacterial sources such as Erwinia chrysanthemi, or it may be genetically engineered in Escherichia coli by recombinant techniques.

[0029] By the conjugation reaction of mPEG and L-asparaginase, 1 to 12 mPEG, preferably 5 to 10 mPEG, more preferably 7 to 10 mPEG, and most preferably 7 to 9 mPEG are covalently bound per L-asparaginase monomer.

[0030] The amount of pegaspargase of the present invention may be present at a concentration (total weight %) of the composition of 2 to 32%, more preferably 5 to 20%, most preferably 6 to 14%.

[0031] The lyophilization process described herein is novel and unique in terms of the optimal amount of excipients utilized in the process. The excipients of the present invention provide physical and chemical stability and biological activity to the compositions of the present invention during storage. The excipients of the present invention also allow for the design of short and economical lyophilization cycles to obtain the products of the present invention. The compositions of the present invention, including pegaspargase and the excipients described herein, exhibit synergistic effects.

[0032] The compositions of the present invention include a cryoprotectant. The cryoprotectant can be selected from sugars, polyols, polymers, and amino acids. More preferably, the cryoprotectant of the present invention is a sugar. Most preferably, the cryoprotectant of the present invention is sucrose. The cryoprotectant may be present in an amount ranging from 9 to 91%, more preferably from 20 to 60%, and most preferably from 32 to 41% of the compositions of the present invention. Without being limited by theory, the compositions of the present invention contemplate a cryoprotectant that can act as both a cryoprotectant and a cryoprotectant to reduce excipient burden during cycling. It can also act as a stabilizer.

[0033] The compositions of the present invention comprise a bulking agent selected from the group consisting of sugars, polyols, polymers, and amino acids, preferably an amino acid selected from the group consisting of glycine, histidine, and arginine, preferably glycine. The bulking agent of the present invention may be present in an amount ranging from 1 to 78%, more preferably from 20 to 60%, and most preferably from 38 to 50% of the compositions of the present invention.

[0034] The compositions of the present invention comprise a buffering agent. The buffering agent may be selected from the group consisting of phosphate buffers such as sodium phosphate buffer (sodium dihydrogen phosphate-disodium hydrogen phosphate) and potassium phosphate buffer (potassium dihydrogen phosphate-dipotassium hydrogen phosphate), TRIS, and citrate buffers. Preferably, the compositions of the present invention comprise a phosphate buffer. The pH of the product before lyophilization and after reconstitution of the lyophilized product may be between 6 and 8. The buffering agent of the present invention may be present in the range of 3 to 33% of the compositions of the present invention, more preferably between 3 and 15%, and most preferably between 4 and 6%.

[0035] The compositions of the present invention may optionally contain a salt selected from the group consisting of sodium chloride and potassium chloride, preferably sodium chloride. The amount of salt in the composition may range from 0 to 40%, preferably 0 to 10%, more preferably 0 to 0.5% of the composition of the present invention. Without being limited by theory, the compositions of the present invention are characterized as being low in salt or salt-free, i.e., unlike prior art compositions, the compositions of the present invention may contain very low amounts of salt or may be salt-free.

[0036] The compositions of the present invention preferably have an osmolality in the range of 250 to 600 mOsm / Kg, more preferably 250 to 500 mOsm / Kg, and most preferably 250 to 450 mOsm / Kg.

[0037] Freeze drying process The lyophilization process is excipient and active ingredient specific and would require process development for each composition. Furthermore, prior art processes are time consuming, require high excipient usage rates, and are uneconomical.

[0038] The freeze-drying process of the present invention formulates the active pharmaceutical ingredient so that the resulting freeze-dried product has the following characteristics: a. A smooth solid substance that does not stick to the sides of the vial b. Stable moisture content c. Extending storage period (room temperature) d. Easily dissolves upon reconstitution to give a clear solution e. Protein activity is not impaired f. No change in protein structure g. pH is maintained h. The reconstituted solution is within an acceptable osmolality range for parenteral administration

[0039] The lyophilization cycle consists of three main steps: freezing, primary drying, and secondary drying, with an optional annealing step between freezing and primary drying. Each of these steps has been optimized for the compositions of the present invention. Furthermore, it is anticipated that the process described here can also be applied to similar compositions containing pegaspargase as an active ingredient.

[0040] The total time for the freeze-drying step of the present invention is preferably 2880 minutes (48 hours) to 5790 minutes (96.5 hours), more preferably 3120 minutes (52 hours) to 4980 minutes (83 hours), and most preferably 3120 minutes (52 hours) to 4200 minutes (70 hours). The freeze-drying step of the present invention preferably involves temperature fluctuations from -60°C to 30°C, more preferably from -50°C to 30°C, and most preferably from -40°C to 25°C. The pressure fluctuation for the freeze-drying step of the present invention is preferably 0.037 Torr to 760 Torr.

[0041] Before freeze drying The concentration of pegaspargase present in the composition prior to lyophilization is preferably in the range of 4 to 25% of the composition, more preferably in the range of 6 to 20%, and most preferably in the range of 8 to 16%.

[0042] The fill volume before freeze-drying is preferably in the range of 0.5 to 5 ml, more preferably in the range of 0.5 to 4 ml, and most preferably in the range of 0.5 to 3 ml.

[0043] Based on the fill volume and the volume after reconstitution, the appropriate concentration of additives (if any) should be added to achieve the desired concentration of additives after reconstitution of the lyophilized product prior to administration.

[0044] Freeze-drying cycle Step-1-Freezing In the freeze-drying step of the present invention, the minimum temperature in the freezing step is preferably -10°C to -60°C, more preferably -20°C to -50°C, and most preferably -35°C to -45°C. The total time for the freezing step in the freeze-drying step of the present invention is preferably 150 to 500 minutes, more preferably 200 to 400 minutes, and most preferably 240 to 350 minutes. The time required to reach the minimum freezing temperature in the freezing step of the freeze-drying step of the present invention is preferably 20 to 180 minutes, more preferably 30 to 120 minutes, and most preferably 45 to 90 minutes. Furthermore, the holding time at the minimum freezing temperature in the freezing step of the freeze-drying step of the present invention is preferably 120 to 480 minutes, more preferably 250 to 360 minutes, and most preferably 200 to 300 minutes.

[0045] Step 2 - Primary drying In the freeze-drying process of the present invention, the starting temperature of the primary drying step is preferably 10°C to -50°C, more preferably 0°C to -45°C, and most preferably -30°C to -40°C. The total time for the primary drying step of the freeze-drying process of the present invention is preferably 35 to 80 hours, more preferably 40 to 75 hours, and most preferably 50 to 60 hours. The time required to reach the starting temperature of the primary drying step of the freeze-drying process of the present invention is preferably 100 to 1000 minutes, more preferably 250 to 500 minutes, and most preferably 300 to 400 minutes. The pressure at the start of the primary drying step of the freeze-drying process of the present invention is preferably 50 mTorr to 200 mTorr. The maximum temperature at the end of the primary drying step of the freeze-drying process of the present invention is preferably 5°C to 25°C, more preferably 8°C to 22°C, and most preferably 10°C to 20°C. The hold time at the highest temperature in the primary drying step of the freeze-drying process of the present invention is preferably 5 to 72 hours, more preferably 8 to 24 hours, and most preferably 10 to 14 hours. The pressure at the end of the primary drying step of the freeze-drying process of the present invention is preferably 37 mTorr to 112 mTorr, more preferably 50 mTorr to 90 mTorr, and most preferably 60 mTorr to 80 mTorr.

[0046] Furthermore, the primary drying step of the freeze-drying process of the present invention may include one or more intermediate drying steps. The temperature of the intermediate drying step of the freeze-drying process of the present invention is preferably -5°C to 15°C, more preferably 0°C to 10°C, and most preferably 3°C to 7°C. The retention time of the intermediate drying step of the freeze-drying process of the present invention is preferably 2 to 24 hours, more preferably 5 to 12 hours, and most preferably 8 to 10 hours. The pressure of the intermediate drying step of the freeze-drying process of the present invention is preferably 75 mTorr to 200 mTorr, and most preferably 100 mTorr to 120 mTorr.

[0047] Step 3: Secondary drying At the end of the primary drying cycle, the dried powder typically contains 10% residual moisture, which must be removed in a secondary cycle. This is the final cycle in the freeze-drying process, which removes the unfrozen water, i.e., the water associated with the amorphous state, and further dries the product to reduce the residual moisture content.

[0048] In the freeze-drying process of the present invention, the temperature in the secondary drying step is preferably 10°C to 37°C, more preferably 15°C to 35°C, and most preferably 20°C to 30°C. The total time for the secondary drying step in the freeze-drying process of the present invention is preferably 3 to 24 hours, more preferably 4 to 16 hours, and most preferably 4 to 7 hours, and the holding time for the secondary drying step in the freeze-drying process of the present invention is preferably 3 to 24 hours, more preferably 4 to 16 hours, and most preferably 4 to 7 hours. The pressure in the secondary drying step in the freeze-drying process of the present invention is preferably 37 mTorr to 50 mTorr.

[0049] After freeze drying The reconstitution volume per vial after lyophilization can be 1 to 5.5 mL depending on the desired dose after reconstitution and the initial concentration of the sample before lyophilization. After reconstitution of the lyophilized product to the required volume, the concentration of pegaspargase is in the range of 750 ± 20% IU / mL.

[0050] Purpose In another aspect of the present invention, the compositions of the present invention have been found to be stable for extended periods of time despite temperature fluctuations that occur during handling and transportation. This is because the products are stable for significant periods of time not only at room temperature but also at 30°C and 37°C. Without being limited by theory, it is proposed that optimal use of the various components in the above ratios maintains the stability of the composition during and after lyophilization, resulting in a more stable product. The compositions of the present invention can produce lyophilized products that maintain their physical integrity, biological activity, and chemical stability.

[0051] Furthermore, the compositions of the present invention contain pegaspargase with a purity of greater than 95% after lyophilization, which has been shown to provide good stability and minimize degradation under both accelerated and real-time stability conditions.

[0052] Additionally, the compositions of the present invention are synergistic in that when the components are combined and formulated in accordance with the principles herein, a composition is obtained that has adequate activity and stability during storage.

[0053] Advantages of the Composition of the Invention 1. The compositions of the present invention provide desirable mechanical support to the solid material structure, thereby imparting stability to the pegaspargase composition and enhancing its ability to withstand the stresses of the freeze-drying process, resulting in a shelf-stable product.

[0054] 2. The process of the present invention significantly reduces the freeze-drying time compared to other prior art processes, thereby reducing freeze-dryer operation time by nearly two days, improving equipment utilization and making the process more economically effective. The freeze-drying process optimized for the novel pegaspargase composition of the present invention was completed in less than three days, a significant improvement over freeze-drying processes that take approximately five days (112.5 hours).

[0055] 3. In the composition of the present invention, the freeze-dried pegaspargase composition can be free of salt or low in salt, which has advantages in terms of eutectic temperature and glass transition temperature.

[0056] 4. The novel composition described in this invention creates a shelf-stable product at higher temperatures. The 18-month stability of lyophilized pegaspargase at 30°C and 3 months at 37°C clearly demonstrates improved thermal stability over liquid formulations. Physical, chemical, and biological degradation of the target composition is minimized under both accelerated and real-time storage conditions. This is especially important for developing countries, as the novel lyophilized pegaspargase composition can tolerate temperature changes during transportation and handling.

[0057] 5. The freeze-dried pegaspargase compositions of the present invention are created simultaneously with the freeze-drying process, reducing the stress of freeze-drying conditions on the product. The compositions of the present invention have high pegaspargase purity and are free of degradants that are often produced as a result of the stress of protein freeze-drying, resulting in reduced immunogenicity of the product (due to the presence of product-associated impurities, primarily degradants). Furthermore, the present invention does not undergo stress-induced aggregation and the generation of higher molecular weight species, as observed with other prior art products, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It should be noted that the liquid compositions of prior art products did not exhibit the presence of high molecular weight species. The novel freeze-dried pegaspargase compositions developed using a novel freeze-drying process corresponding to the compositions of the present invention do not exhibit the presence of high molecular weight species.

[0058] 6. The present invention provides a freeze-dried product with high storage stability at an optimal cost based on the optimal amount of excipients and the optimal freeze-drying process.

[0059] The present invention is illustrated herein by examples. The examples provide a description of the compositions of the present invention and the protection of pegaspargase during lyophilization and storage. The examples are illustrative of one embodiment of the present invention and should not be construed as limiting in any way. [Example]

[0060] As has been repeatedly stated, the freeze-drying process to obtain a shelf-stable product depends on the formulation composition, which determines the fate of the product after freeze-drying, and therefore the freeze-drying cycle and composition must be developed simultaneously. Examples 1 and 2 show in detail the interdependence between the freeze-drying process and the composition.

[0061] Example 1: Effect of excipients 1.1 Cryoprotectants Pegaspargase bulk was buffer-exchanged with 50 mM sodium phosphate buffer (pH 7.4). The bulk (drug substance) was formulated with various weight percents (of the composition) of cryoprotectants, namely, sucrose and trehalose. One mL of the formulated pegaspargase stock solution was filled into sterile, depyrogenated USP Type I 2 mL glass vials recommended for parenteral administration and half-closed with 13 mm gray bromobutyl-coated rubber stoppers. The half-closed vials were subjected to a lyophilization process.

[0062] The freeze-drying process involved an initial freeze at -40°C over 1 hour at a freeze rate of 1.08°C / min, followed by a 3-hour hold. The primary drying step involved ramping the temperature to -5°C at 0.028°C / min under 112 mTorr and holding at that temperature for 6 hours. The temperature was then ramped to 0°C at 0.006°C / min and held at 0°C for 6 hours at 112 mTorr. Finally, the temperature was ramped to 20°C at 0.03°C / min and held at 112 mTorr for 5 hours. The secondary drying step involved further reducing the pressure to 37 mTorr, ramping the temperature to 25°C at 0.17°C / min and holding at 25°C for 5 hours. The total freeze-drying time was 76.5 hours.

[0063] After the freeze-drying process was completed, the vials were completely closed by moving the shelves upward. Sterile nitrogen gas was then introduced into the freeze-drying chamber to release the pressure. The freeze-dried vials were then sealed with 13 mm flip-off seals and subjected to analytical characterization. The freeze-dried products were measured for solid structure, reconstitution time, clarity after reconstitution, and relative activity (relative to the pre-lyophilized sample). The results of the freeze-drying process for various compositions of pegaspargase with different cryoprotectants are shown in Table 1.

[0064] [Table 1]

[0065] The solid structure of formulated pegaspargase in the presence of various cryoprotectants at different ratios in the composition of the lyophilized product was not satisfactory, and therefore the addition of bulking agents was required to achieve satisfactory results.

[0066] 1.2 Bulking agents Pegaspargase bulk was buffer-exchanged with 50 mM sodium phosphate buffer (pH 7.4). The bulk (drug substance) was formulated with various weight percent (of the composition) of fillers, namely, mannitol and glycine. One mL of the formulated pegaspargase stock solution was filled into sterile, depyrogenated USP Type I 2 mL glass vials recommended for parenteral administration and half-closed with 13 mm gray bromobutyl-coated rubber stoppers. The half-closed vials were subjected to a lyophilization process.

[0067] The freeze-drying process involved an initial freeze at -40°C over 1 hour at a freezing rate of 1.08°C / min, followed by a 3-hour hold. The primary drying step involved ramping the temperature to -35°C at 0.028°C / min under 112 mTorr pressure and holding at that temperature for 10 hours. The temperature was then ramped to 5°C at 0.11°C / min and held at 5°C for 9 hours under 112 mTorr pressure. Finally, the temperature was ramped to 15°C at 0.13°C / min and held at 15°C for 12 hours under a vacuum of 75 mTorr. The secondary drying step involved further reducing the pressure to 37 mTorr, ramping the temperature to 25°C at 0.33°C / min and holding at that temperature for 5 hours. The total freeze-drying time was 53 hours.

[0068] After the freeze-drying process was completed, the vials were completely closed by moving the shelves upward. Sterile nitrogen gas was then introduced into the freeze-drying chamber to release the pressure. The freeze-dried vials were then sealed with 13 mm flip-off seals and subjected to analytical characterization. The freeze-dried products were measured for solid structure, reconstitution time, clarity after reconstitution, and relative activity and purity (relative to the pre-lyophilized sample). The results of the freeze-drying process for various compositions of pegaspargase with different bulking agents are shown in Table 2.

[0069] [Table 2]

[0070] The structure of the solid material is shown in Figure 1. As is evident from this data set, the bulking agent glycine contributes significantly to the structure of the solid material and maintains the activity within an acceptable range (600 IU / mL to 900 IU / mL). The structure of the solid material without the bulking agent in this lyophilization process is pharmaceutically acceptable, but the activity and purity of the pegaspargase are very low. When mannitol is used as a bulking agent, the activity and purity are maintained, but this lyophilization process does not produce a good solid material structure.

[0071] 1.3 The effects of salt Pegaspargase bulk was buffer-exchanged into 50 mM sodium phosphate buffer (pH 7.4) and formulated with sucrose (cryoprotectant / cryoprotectant), varying amounts of bulking agent (glycine), and varying amounts (% by weight of composition) of salt. 2 ml of the formulated bulk was filled into sterile, depyrogenated USP Type I 5 mL glass vials recommended for parenteral administration and half-closed with 20 mm gray bromobutyl-coated rubber stoppers. The half-closed vials were subjected to an optimized lyophilization process.

[0072] The freeze-drying step was carried out at -40°C for 4 hours. The freezing temperature was reached at a freezing rate of 1°C / min. In the primary drying step, the temperature was raised to -35°C at a rate of 0.014°C / min under 112 mTorr pressure and held at that temperature for 10 hours. The temperature was then raised to 5°C at a rate of 0.06°C / min under 112 mTorr pressure and held at 5°C for 9 hours. The pressure was then reduced to 75 mTorr, and the temperature was raised to 15°C at a rate of 0.02°C / min and held for 12 hours. In the secondary drying cycle, the pressure was further reduced to 37 mTorr, and the temperature was raised to 25°C at a rate of 0.33°C / min and held for 5 hours.

[0073] After the freeze-drying process was completed, the vials were completely closed by moving the shelves upward. Sterile nitrogen gas was then introduced into the freeze-drying chamber to release the pressure. The freeze-dried vials were then sealed with 20 mm flip-off seals. The freeze-dried products were reconstituted with 5 mL of water for injection and subjected to analytical characterization. The freeze-dried products were measured for solid structure, reconstitution time, clarity after reconstitution, relative activity (relative to the bulk before freeze-drying), absolute purity (expressed as a percentage as measured by size-exclusion high-performance liquid chromatography (SE-HPLC)), and osmolality. The results of the freeze-drying process for various compositions of pegaspargase are shown in Table 3.

[0074] [Table 3]

[0075] It is clear that the optimized freeze-drying process can be used for low-salt and no-salt compositions without altering important product attributes.

[0076] Example 2: Effect of different freeze-drying cycles on the same composition Pegaspargase bulk was buffer-exchanged with 50 mM sodium phosphate buffer (pH 7.4). The bulk (drug substance) was formulated with sucrose (34.3% cryoprotectant / cryoprotectant in the composition) and glycine (51.5% bulking agent in the composition). 1 ml of the formulated bulk was filled into sterile, depyrogenated USP Type I 2 mL glass vials recommended for parenteral administration and half-closed with 13 mm gray bromobutyl-coated rubber stoppers. The vials were subjected to various lyophilization steps.

[0077] The freezing steps of the various freeze-drying processes were performed for various durations and temperatures. In some cases, a single-step freeze was performed, while in others, a multi-step freeze was performed. In one cycle, the initial freeze was performed at -15°C for 2 hours at a freezing rate of 1.16°C / min. The temperature was then further reduced to -25°C at a freezing rate of 0.33°C / min, and the vials were held there for 3 hours. Finally, the temperature was reduced to -40°C at a freezing rate of 0.5°C / min, and the vials were held there for 2 hours. The total freezing step duration was 8.5 hours. In another cycle, the freeze-drying process was performed at -40°C for 3 hours. The freezing temperature was reached at a freezing rate of 1°C / min. The total freezing step duration was 4 hours. In another cycle, the freeze-drying process was performed at -40°C for 6 hours. The freezing temperature was reached at a freezing rate of 0.5°C / min. The total freezing step duration was 8 hours. In yet another cycle, the freeze-drying process was performed at 40°C for 4 hours. The freezing temperature was reached at a freezing rate of 1°C / min. The total time for the freezing step was 5 hours.

[0078] The primary drying step of the freeze-drying cycle was also varied in terms of temperature, pressure, and time. In one cycle, the primary drying step was ramped to -5°C at a rate of 0.028°C / min under 112 mTorr and held at that temperature for 6 hours. The temperature was then ramped to 0°C at a rate of 0.006°C / min and held at 0°C for 6 hours under 112 mTorr. Finally, the temperature was ramped to 20°C at a rate of 0.03°C / min and held at 20°C for 5 hours under 112 mTorr. The total primary drying step time was 61.5 hours. In another cycle, the temperature was ramped to -5°C at a rate of 0.15°C / min under 112 mTorr and held at that temperature for 14 hours. The temperature was then ramped to 5°C at a rate of 0.06°C / min and held at 5°C for 9 hours under 112 mTorr. Finally, the pressure was reduced to 75 mTorr, and the temperature was increased to 15 °C at a rate of 0.067 °C / min and maintained for 6 hours. The total time for the primary drying step was 38.5 hours. In another cycle of the primary drying step, the temperature was increased to -35 °C at a rate of 0.027 °C / min under 112 mTorr and held at that temperature for 10 hours. The temperature was then increased to 5 °C at a rate of 0.11 °C / min and maintained at 5 °C for 9 hours under 112 mTorr. Finally, the pressure was reduced to 75 mTorr, and the temperature was increased to 15 °C at a rate of 0.067 °C / min and maintained for 12 hours. The total time for the primary drying step was 42.5 hours. In another cycle of the primary drying step, the temperature was increased to -35 °C at a rate of 0.027 °C / min under 112 mTorr and held at that temperature for 10 hours. The temperature was then ramped to 5°C at 0.11°C / min and maintained at 5°C for 9 hours at 112 mTorr. The temperature was then ramped to 10°C at 0.014°C / min and maintained at 10°C for 24 hours at 112 mTorr. Finally, the pressure was reduced to 75 mTorr, and the temperature was ramped to 15°C at 0.033°C / min and maintained for 12 hours. The total time for the primary drying step was 72.5 hours. In another cycle of the primary drying step, the temperature was ramped to -35°C at 0.027°C / min under 112 mTorr and held at that temperature for 6 hours. The temperature was then ramped to 15°C at 0.138°C / min and maintained at 15°C for 9 hours at 112 mTorr.Finally, the pressure was reduced to 75 mTorr over 5 hours and maintained for 12 hours at 15°C. The total time for the primary drying step was 38.5 hours.

[0079] The secondary drying step of the freeze-drying cycle was also varied in terms of temperature, pressure, and time. In one cycle, the pressure in the secondary drying step was reduced to 37 mTorr, and the temperature was increased to 25°C at a rate of 0.16°C / min and maintained for 5 hours. The total time for the secondary drying step was 5.5 hours. In one cycle, the pressure in the secondary drying step was further reduced to 37 mTorr, and the temperature was increased to 25°C at a rate of 0.33°C / min and maintained for 5 hours. The total time for the secondary drying step was 5.5 hours. In yet another cycle, the pressure in the secondary drying step was further reduced to 37 mTorr, and the temperature was increased to 25°C at a rate of 0.33°C / min and maintained for 9 hours. The total time for the secondary drying step was 9.5 hours.

[0080] The completion time of the freeze-drying process varied from 48 to 83.5 hours.

[0081] After the lyophilization process was completed, the vials were completely closed by moving the shelves upward. Sterile nitrogen gas was then introduced into the lyophilization chamber to release the pressure. The lyophilized vials were then sealed with 13 mm flip-off seals and subjected to analytical characterization. The lyophilized products were measured for solid structure, reconstitution time, post-reconstitution clarity, relative activity (relative to the bulk before lyophilization), relative purity (relative to the bulk before lyophilization as measured by size-exclusion high-performance liquid chromatography (SE-HPLC)), and osmolality. The lyophilized products had good and satisfactory solid formation, acceptable reconstitution times (less than 2 minutes), and reconstituted samples were colorless and transparent. However, as shown in Table 4, there was considerable variability in relative activity and purity. This result is expected due to the different stress conditions imposed on the same composition due to variations in the lyophilization process.

[0082] [Table 4]

[0083] Example 3: Illustrative Examples of Compositions of the Present Invention - Low Salt The pegaspargase bulk was buffer-exchanged with 50 mM sodium phosphate buffer (pH 7.4). Sucrose was added as a cryoprotectant and glycine as a bulking agent, and the resulting bulk was diluted to a final concentration of 18751 U / mL. The final formulations contained pegaspargase, sucrose, glycine, monobasic sodium phosphate, dibasic sodium phosphate, and sodium chloride at 13.67% to 7.04%, 39.60% to 36.78%, 47.52% to 44.13%, 0.95% to 0.88%, 4.42% to 4.10%, and 0.47% to 0.43% of the composition, respectively. Two milliliters of the formulated bulk was filled into sterile, depyrogenated USP Type I 5 mL glass vials recommended for parenteral administration and partially closed with 20 mm gray bromobutyl-coated rubber stoppers. The vials were subjected to an optimized freeze-drying process in a semi-closed state.

[0084] The freezing step of the freeze-drying process was carried out at -40°C for 4 hours. The freezing temperature was reached at a freezing rate of 1°C / min. In the primary drying cycle, the temperature was increased to -35°C at a rate of 0.014°C / min under 112 mTorr and held at that temperature for 10 hours. The temperature was then increased to 5°C at a rate of 0.06°C / min and maintained at 5°C for 9 hours under a pressure of 112 mTorr. The pressure was then reduced to 75 mTorr, and the temperature was increased to 15°C at a rate of 0.02°C / min and held for 12 hours. In the secondary drying cycle, the pressure was further reduced to 37 mTorr, and the temperature was increased to 25°C at a rate of 0.33°C / min and held for 5 hours. The total freeze-drying process time was 66.5 hours.

[0085] After the freeze-drying process was completed, the vials were completely closed by moving the shelves upward. Sterile nitrogen gas was then introduced into the freeze-drying chamber to release the pressure. The freeze-dried vials were then sealed with 20 mm flip-off seals. The freeze-dried products were reconstituted with 5 mL of water for injection and subjected to analytical characterization (Table 5).

[0086] [Table 5]

[0087] Figure 2 shows the integrity and purity of pegaspargase before and after lyophilization as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion high-performance liquid chromatography (SE-HPLC).

[0088] The composition and process robustness of the lyophilization cycle were verified on multiple batches. The lyophilized products were measured for solid structure, reconstitution time, clarity after reconstitution, absolute activity, relative activity (relative to the bulk before lyophilization), absolute purity (expressed as a percentage as determined by size-exclusion high-performance liquid chromatography (SE-HPLC)), relative purity (relative to the bulk before lyophilization), osmolality, and moisture content. Product properties met the acceptance criteria shown in Table 6 for three representative batches.

[0089] [Table 6]

[0090] The lyophilized product underwent a 24-month long-term stability test at 5°C ± 3°C and a 6-month accelerated stability test at 25°C ± 2°C / 60% ± 5% relative humidity in accordance with the ICH Quality Guideline (Q1A). The results of the lyophilization of the product pegaspargase at room temperature (5°C ± 3°C) and at 25°C ± 2°C / 60% ± 5% relative humidity are shown in Tables 7 and 8, respectively.

[0091] [Table 7]

[0092] [Table 8]

[0093] The product properties were stable for 24 months at 5°C ± 3°C and for 6 months at 25°C ± 2°C / 60% ± 5% relative humidity, meeting the acceptance criteria throughout the entire period. Furthermore, to evaluate the stability of the lyophilized product at elevated temperatures, a long-term stability study was conducted for 18 months at 30°C ± 2°C / 65% ± 5% relative humidity, and an accelerated stability study was conducted for 3 months at 37°C ± 2°C / 75% ± 5% relative humidity. The results of the lyophilization process for Pegaspargase at various time points at 30°C ± 2°C / 65% ± 5% relative humidity and 37°C ± 2°C / 75% ± 5% relative humidity are shown in Tables 9 and 10, respectively. The product properties were stable for 18 months at 30°C ± 2°C / 65% ± 5% relative humidity and for 3 months at 37°C ± 2°C / 75% ± 5% relative humidity, meeting the acceptance criteria throughout the entire period.

[0094] [Table 9]

[0095] [Table 10]

[0096] Example 4: Illustrative Examples of Compositions of the Present Invention - Salt-Free Pegaspargase bulk was buffer-exchanged with 50 mM sodium phosphate buffer (pH 7.4). The required concentrated bulk was formulated using sucrose as a cryoprotectant / cryoprotectant and glycine as a bulking agent, diluted to a final concentration of 18751 U / mL. The final formulations consisted of pegaspargase, sucrose, glycine, monobasic sodium phosphate, and dibasic sodium phosphate, comprising 12.57% to 9.60%, 38.71% to 37.43%, 46.45% to 44.92%, 0.93% to 0.90%, and 4.32% to 4.18% of the composition, respectively. 2 mL of the formulated bulk was filled into sterile, depyrogenated USP Type I 5 mL glass vials recommended for parenteral administration and partially closed with 20 mm gray bromobutyl-coated rubber stoppers. The vials were subjected to an optimized freeze-drying process in a semi-closed state.

[0097] The freezing step of the freeze-drying process was carried out at -40°C over a period of 4 hours. The freezing temperature was reached at a freezing rate of 1°C / min. In the primary drying cycle, the temperature was increased to -35°C at a rate of 0.014°C / min under a pressure of 112 mTorr and held at that temperature for 10 hours. The temperature was then increased to 5°C at a rate of 0.06°C / min and held at 5°C for 9 hours under a pressure of 112 mTorr. The pressure was then reduced to 75 mTorr, and the temperature was increased to 15°C at a rate of 0.02°C / min and held for 12 hours. In the secondary drying cycle, the pressure was further reduced to 37 mTorr, and the temperature was increased to 25°C at a rate of 0.33°C / min and held for 5 hours. The total freeze-drying process time was 66.5 hours.

[0098] After the freeze-drying process was completed, the vials were completely closed by moving the shelves upward. Sterile nitrogen gas was then introduced into the freeze-drying chamber to release the pressure. The freeze-dried vials were then sealed with 20 mm flip-off seals. The freeze-dried products were reconstituted with 5 mL of water for injection and submitted for analytical characterization (Table 11).

[0099] [Table 11]

[0100] The composition and process robustness of the lyophilization cycle were verified on multiple batches. The lyophilized product was measured for solid structure, reconstitution time, clarity after reconstitution, absolute activity, relative activity (relative to the bulk before lyophilization), absolute purity (expressed as a percentage as determined by size-exclusion high-performance liquid chromatography (SE-HPLC)), relative purity (relative to the bulk before lyophilization), osmolality, and moisture content. Product properties met the acceptance criteria shown in Table 12 for three representative batches.

[0101] [Table 12]

[0102] The salt-free freeze-dried product was subjected to long-term stability tests at 5°C±3°C, 25°C±2°C / 60%±5%, 30°C±2°C / 65%±5% relative humidity, and 37°C±2°C / 75%±5% relative humidity. The data for one month are shown in Table 13, and the data for three months are shown in Table 14.

[0103] [Table 13]

[0104] [Table 14]

[0105] Example 5: Comparison of the composition of the present invention with the prior art 5.1 Prior Art Liquid Compositions vs. Solid Compositions of the Invention Pegaspargase reported in the prior art is generally presented as a liquid composition and lacks shelf stability for longer periods and at higher temperatures. The present invention discloses a freeze-dried composition that is shelf-stable at various temperatures. Conventional pegaspargase has been known to lack long-term stability. The present invention overcomes this limitation and provides a shelf-stable composition. Prior art products significantly deteriorate within three months at 25°C ± 2°C / 60% ± 5% relative humidity. As shown in Figure 3, the deterioration of the liquid formulation is evident by the presence of multiple bands in the liquid composition at 25°C ± 2°C / 60% ± 5% relative humidity when analyzed by SDS-PAGE (Figure 3(A)). The present invention provides a shelf-stable formulation that is stable at 25°C ± 2°C / 60% ± 5% relative humidity, 30°C ± 2°C / 65% ± 5% relative humidity, and 37°C ± 2°C / 75% ± 5% relative humidity. The stability of the composition of the present invention is evident by the presence of a single diffuse band of the appropriate molecular weight, as shown in Figure 3(B).

[0106] 5.2 Prior Art Solid Compositions vs. Solid Compositions of the Invention While the prior art discloses compositions with high shelf-stability, they are limited by the occurrence of high molecular weight aggregates in the lyophilized product. The present disclosure utilizes an inventive process and improved composition, resulting in the absence of additional aggregates / high molecular weight impurities (see Figure 4). Figure 4 shows an SDS-PAGE analysis of a prior art lyophilized composition according to the present invention. The SDS-PAGE gel confirms the presence of high molecular weight impurities, as evidenced by protein Coomassie staining (Figure 4(A)) and PEG iodine staining (Figure 4(B)). Furthermore, Western blot analysis using anti-asparaginase antibodies (Figure 4(C)) and anti-PEG antibodies (Figure 4(B)) confirmed the presence of product-related impurities. This further highlights the synergistic effect of the lyophilization process and the formulation composition. It is noteworthy that the presence of high molecular weight species was not observed in the liquid composition of the prior art product.

Claims

1. A freeze-dried composition with optimal storage stability comprising pegaspargase, a cryoprotectant, a bulking agent, a buffer, and optionally a pharmaceutically acceptable excipient.

2. the peg asparaginase is a pegylated asparaginase comprising a polyalkylene oxide group covalently attached to the asparaginase by a linker; the polyalkylene oxide is a monomethoxypolyethylene glycol (mPEG) having a molecular weight of preferably 4 to 6 kDa, more preferably 4.5 to 5.5 kDa, and most preferably 4.8 to 5.2 kDa, and is covalently attached to one or more primary amine groups of the L-asparaginase via an amide bond through a succinic acid linker through conjugation; The coupling reaction of mPEG with L-asparaginase results in covalently binding 1 to 12 mPEG per L-asparaginase monomer, preferably 5 to 10 mPEG per L-asparaginase monomer, more preferably 7 to 10 mPEG per L-asparaginase monomer, and most preferably 7 to 9 mPEG per L-asparaginase monomer.

10. The composition of claim 1.

3. 3. The composition of claim 2, wherein the L-asparaginase is derived from a bacterial source selected from the group consisting of Escherichia coli or Erwinia chrysanthemi, or is obtained by recombinantly engineered Escherichia coli.

4. 2. The composition of claim 1, wherein the amount of pegaspargase in the composition is from 2 to 32%, more preferably from 5 to 20%, and most preferably from 6 to 14% of the composition.

5. the cryoprotectant is selected from the group consisting of sugars, polyols, polymers, and amino acids, preferably sugars; The sugar is sucrose 2. The composition of claim 1, wherein the cryoprotectant is present in the range of 9 to 91%, preferably 20 to 60%, most preferably 32 to 41% of the composition.

6. the bulking agent is selected from the group consisting of sugars, polyols, polymers, and amino acids, preferably amino acids; the amino acid is selected from the group consisting of glycine, histidine, and arginine, preferably the amino acid is glycine; The bulking agent is present in the range of 1 to 78% of the composition, more preferably 20 to 60%, and most preferably 38 to 50%.

10. The composition of claim 1.

7. The buffer is selected from the group consisting of a phosphate buffer, a sodium phosphate buffer (sodium dihydrogen phosphate-disodium hydrogen phosphate), a potassium phosphate buffer (potassium dihydrogen phosphate-dipotassium hydrogen phosphate), TRIS, and a citrate buffer, and is preferably a phosphate buffer; The buffering agent ranges from 3 to 33% of the composition, more preferably from 3 to 15%, and most preferably from 4 to 6%.

10. The composition of claim 1.

8. 10. The composition of claim 1, wherein the pharmaceutically acceptable excipient is a salt.

9. the salt is selected from the group consisting of sodium chloride and potassium chloride, preferably sodium chloride; The amount of said salt in said composition ranges from 0 to 40%, preferably from 0 to 10%, more preferably from 0 to 0.5% of said composition; 9. The composition of claim 8.

10. the pH of the product before lyophilization and after reconstitution of the lyophilized product is between 6 and 8; The osmolality is preferably in the range of 250 to 600 mOsm / Kg, more preferably 250 to 500 mOsm / Kg, and most preferably 250 to 450 mOsm / Kg.

10. The composition of claim 1.

11. a. Freezing step, b. an optional annealing step; c. A primary drying step, and d. A secondary drying step; A composition preparation process for preparing the composition of claim 1.

12. the total time for the freeze-drying step is preferably between 2880 minutes (48 hours) and 5790 minutes (96.5 hours), more preferably between 3120 minutes (52 hours) and 4980 minutes (83 hours), and most preferably between 3120 minutes (52 hours) and 4200 minutes (70 hours); the temperature change is from -60°C to 30°C, more preferably from -50°C to 30°C, and most preferably from -40°C to 25°C; The pressure change is preferably from 0.037 Torr to 760 Torr.

12. A process for preparing the composition according to claim 11.

13. the freezing step is carried out at a minimum temperature of -10°C to -60°C, more preferably -20°C to -50°C, most preferably -35°C to -45°C; the total time is preferably from 150 to 500 minutes, more preferably from 200 to 400 minutes, and most preferably from 240 to 350 minutes; The minimum freezing temperature is preferably 20 to 180 minutes, more preferably 30 to 120 minutes, and most preferably 45 to 90 minutes; The holding time at the minimum freezing temperature is preferably 120 to 480 minutes, more preferably 250 to 360 minutes, and most preferably 200 to 300 minutes. The process of claim 11.

14. the primary drying is at 10°C to -50°C, more preferably 0°C to -45°C, and most preferably -30°C to -40°C; the total time is preferably from 35 to 80 hours, more preferably from 40 to 75 hours, and most preferably from 50 to 60 hours; the time required to reach the initiation temperature is preferably between 100 and 1000 minutes, more preferably between 250 and 500 minutes, and most preferably between 300 and 400 minutes; The starting pressure is preferably between 50 mTorr and 200 mTorr; the maximum temperature at the end of the primary drying step is preferably from 5°C to 25°C, more preferably from 8°C to 22°C, and most preferably from 10°C to 20°C; The holding time at the highest temperature in the primary drying step of the freeze-drying process of the present invention is preferably 5 to 72 hours, more preferably 8 to 24 hours, and most preferably 10 to 14 hours; The pressure at the end of the primary drying step of the freeze-drying process of the present invention is preferably 37 mTorr to 112 mTorr, more preferably 50 mTorr to 90 mTorr, and most preferably 60 mTorr to 80 mTorr. The process of claim 11.

15. said secondary drying step being at a temperature of from 10°C to 37°C, more preferably from 15°C to 35°C, most preferably from 20°C to 30°C; the total time is preferably 3 to 24 hours, more preferably 4 to 16 hours, and most preferably 4 to 7 hours; the holding time of the secondary drying step of the freeze-drying is preferably 3 to 24 hours, more preferably 4 to 16 hours, and most preferably 4 to 7 hours; The pressure is preferably 37 mTorr to 50 mTorr. The process of claim 11.

16. 12. A freeze-dried composition obtained from the process of claim 11.

17. The reconstitution volume per vial is 1 to 5.5 mL after lyophilization, preferably in the range of 4 to 25% of the composition before lyophilization, more preferably in the range of 6 to 20%, and most preferably in the range of 8 to 16%, with a final pegaspargase concentration in the range of 750±20% IU / ml.

17. The composition of claim 1 or 16.

Images