Sanjay Bandyopadhyay, PhD, is vice-president in the Department of Biotechnology, Zydus Research Centre, Cadila Healthcare Ltd., Ahmedabad, Gujarat, India

Sanjay Bandyopadhyay

	Submitted: Dec. 22, 2018.
	Accepted: Jan. 8, 2019.

	Pegylation is a well-known technology used to increase residence time of L-asparaginase in blood circulation and to reduce hypersensitivity reactions; however, it is important to address manufacturing problems associated with the shorter shelf-life of pegylated L-asparaginase (pegaspargase) upon long-term storage in the form of a solution. Exposure to the sudden excursions encountered during storage and shipping may affect stability of the pegaspargase drug product material. In this article, the effect of freeze-thawing and high temperature on the stability of pegaspargase protein was studied. Differences in the degradation pathways for the storage conditions were identified, and manufacturing issues associated with the degradation are discussed. It was observed that pegaspargase follows different degradation pathways when exposed to freeze-thawing and high temperature stress. These differences in the degradation pathways have different implications on the manufacturing process. 

L-asparaginase has been extensively investigated for the treatment of acute lymphoblastic leukemia (ALL) (1). The role of L-asparaginase present in guinea pig serum in the reduction of lymphoma was first studied by Broome, and the inhibitory effect was attributed to depletion of asparagine due to the presence of L-asparaginase in the serum (2). L-asparaginase enzymatically cleaves amino acid L-asparagine into aspartic acid and ammonia. Depletion of L-asparagine in blood serum results in inhibition of protein-synthesis, DNA-synthesis, and RNA-synthesis, especially in leukemic blasts that are not able to synthesize L-asparagine and thus undergo apoptosis (3). Normal cells, in contrast, are capable of synthesizing L-asparagine and are less affected by its rapid withdrawal during treatment with the enzyme L-asparaginase (4).

-asparaginase: Kidrolase, EUSA Pharma; Elspar, Ovation Pharmaceuticals; Crasnitin, Bayer; Leunase, Sanofi-Aventis; Asparaginase medac, Kyowa Hakko) has been used in the treatment of ALL (5). Although L-asparaginase is an effective antineoplastic agent used in chemotherapy of ALL, development of antibodies against L-asparaginase and hypersensitivity reactions may lead to discontinuation of the treatment (6). Such adverse events along with the other toxicities like thrombosis, pancreatitis, hyperglycemia, and hepatotoxicity led to the development of alternative sources of L-asparaginase (7). The development of L-asparaginase from Erwinia chrysanthemi (Erwinase, EUSA Pharma) is an outcome of such efforts (8). The disadvantage of frequent intramuscular injections and the adverse events of hypersensitivity reactions led to development of a pegylated version of native 

-asparaginase (polyethylene glycol [PEG]-asparaginase: Oncaspar, Enzon Pharmaceuticals Inc) (9). Oncaspar is an asparagine-specific enzyme indicated as a component of a multi-agent chemotherapeutic regimen for treatment of patients with first line ALL and hypersensitivity to asparaginase. Oncaspar is available as a solution in a single-use glass vial with the strength of 3750 IU per 5 mL of solution. The recommended dose of Oncaspar is 2500 IU/m

intramuscularly or intravenously. Oncaspar is not recommended to be used when it is frozen or stored at room temperature 15–25 °C (59–77 °F) for more than 48 hours.

	L-asparaginase is a homotetrameric enzyme comprised of four identical subunits with a mass of 34,592 Da coupled by weak, non-covalent, largely hydrophobic interactions (10). The tetrameric structure of the L-asparaginase enzyme is required for enzymatic activity (11). Pegaspargase (pegylated L-asparaginase) is a covalent conjugate of 

-derived L-asparaginase with monomethoxypolyethylene glycol (mPEG) succinimidyl succinate (PEG-SS; molecular weight of 5000 Da). Approximately 69 to 82 molecules of mPEG are linked to L-asparaginase. The succinyl linker between PEG and L-asparaginase contains an ester linkage that can lead to hydrolytic removal of the PEG moiety from the PEG-protein conjugate (12). The release of PEG from the protein molecule (i.e., depegylation) upon storage in the form of solution leads to a shorter shelf-life. Also, stability of the protein molecule can be affected by chemical as well as physical factors (13). Proteins can be denatured readily by stresses encountered in solution, or in a frozen or dried state (14). It is also important to know the effect of sudden excursions encountered during shipping of the drug product material, which may affect stability of the product. Drug product may be exposed to lower or higher temperature from its real-time storage conditions during shipment. It is known that L-asparaginase shows changes in its structural and biological properties upon repeated freeze-thaw. In this article, the effect of freeze-thaw stress and high temperature on the stability of pegaspargase (pegylated 

-derived L-asparaginase) has been investigated; manufacturing issues associated with these degradation pathways are discussed. 

L-asparaginase was procured in the form of lyophilized powder. PEG of 5000 Da size was procured in the activated form. Pegaspargase was prepared by conjugating (pegylation) multiple units of 5 kDa activated PEG (m-PEG-N-hydroxysuccinimidyl ester) at different sites of the α-NH

group of Lysine residues of the L-asparaginase enzyme using the pegylation techniques known in the art (15, 16). Pegylation occurs in all four subunits at the specified sites of the enzyme. The pegylated L-asparaginase (crude solution) was purified to remove excess free PEG using column chromatography (AKTA system, GE Healthcare). Buffer exchange of the purified pegylated L-asparaginase protein solution was performed in the desired formulation media (with final concentration of 5.58 mg of dibasic sodium phosphate, 1.20 mg of monobasic sodium phosphate, and 8.50 mg of sodium chloride in 1 mL of water for injection) by membrane ultrafiltration and diafiltration steps. Ultrafiltration and diafiltration were performed in a controlled manner at room temperature with 50 kDa molecular weight cut off membrane by using a tangential flow filtration system (Sartorius Stedim Biotech). At the end of buffer exchange, the protein solution was concentrated through an ultrafiltration step, and the final concentration was adjusted to approximately 8.8 mg/mL. The protein solution was finally filtered through a 0.2-µm sterile filter (Sartopore 2, Sartorius Stedim Biotech). The final formulation composition was maintained to be the same as that of the commercial product Oncaspar. All the excipients for the final formulation (i.e., dibasic sodium phosphate, monobasic sodium phosphate, and sodium chloride) were obtained from Merck, Germany. 

Freeze-thawing and exposure to high temperature. 

In order to check the effect of freezing and thawing, pegaspargase solution was aliquoted with 500 µL solution in cryo-vials of 1-mL capacity (Nunc CryoTube vials, Thermo Scientific). Samples were frozen at or below –20 °C in a deep freezer (Thermo Electron Corporation, Model No.: ULT1740-3-V40). Thawing was performed at room-temperature rapidly until the frozen mass was converted into the liquid solution. In order to check the effect of high temperature (forced degradation due to heat), formulated pegaspargase protein solution was filled in 2-mL glass vials (borosilicate USP Type I clear flint glass, Schott) with coated stoppers (fluorinated polymer-coated butyl rubber stopper, West Pharmaceuticals) and sealed with flip-off seals (aluminium seals with flip-off plastic cap, West Pharmaceuticals). Samples were exposed to a controlled high temperature of 40 °C ± 5 °C with 75% ± 5% relative humidity (RH) and 25 °C ± 5 °C with 60% ± 5% RH in the stability chamber (Thermo). Samples were withdrawn at different time intervals and stored between +2 °C and +8 °C before analysis.

Samples were analyzed by high-performance size exclusion chromatography (HP-SEC) using two different detectors: an ultraviolet (UV) detector and a refractive index (RI) detector. The RI detector was used to detect an increase in free PEG due to exposure of pegaspargase to freeze-thawing and high temperature, if any. Polypeptide profile of pegaspargase protein was also evaluated by analyzing samples using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

	HP-SEC of pegaspargase was performed on a high-performance liquid chromatography system (LC 2010-CHT series, Shimadzu) equipped with a YMC Pack Diol-300 column (8.0 × 300 mm; 5 µm). Before injecting the sample, the column was pre-equilibrated with buffer containing 90% 50 mM sodium phosphate (pH 6.8) with 300 mM sodium chloride and 10% 2-propanol at a flow rate of 0.5 mL/min under an oven temperature of 25 °C. After equilibrating the column, 10 µg of sample was injected and analyzed in isocratic mode at a flow rate of 0.5 mL/min, and chromatographic separation was monitored at 214 nm with UV detection or using the RI detector to monitor free PEG in the samples. 

	The polypeptide profile of pegaspargase was obtained under non-reducing conditions. SDS-PAGE was carried out essentially in accordance with the Laemmli method (1970) (17). For non-reducing SDS-PAGE, 4–20% gradient polyacrylamide gel (Bio-Rad) was used. A total of 10 µg of pegaspargase protein was loaded for different test samples on the gel. After electrophoresis, protein bands were developed by either Coomassie R250 (Merck) staining or barium iodide staining using barium chloride (Merck) and iodine (Merck). Coomassie R250 is known to be more sensitive compared to Coomassie G250 and is commonly used to stain the protein molecules during SDS-PAGE. Barium iodide forms a complex with PEG and is a well-known technique to stain PEG after gel electrophoresis. In the present work, degradation of pegylated L-asparaginase molecule (aggregation or fragmentation) upon exposure to freezing and thawing or high temperature was detected using Coomassie R250 staining, while the presence of free PEG was detected by barium iodide staining. 

	The effects of freeze-thaw and high temperature on pegaspargase protein were investigated, and differences in the degradation pathways were evaluated. 

Effect of freeze-thaw on stability of pegaspargase. 

Pegaspargase protein formulated at about 8.8 mg/mL concentration in 49-mM sodium phosphate buffer in presence of 146-mM sodium chloride was exposed to freeze-thaw stress as described in the previous section (Materials and methods). After thawing, samples were analyzed by HP-SEC and SDS-PAGE. 

	Chromatographic profiles obtained with the samples of pegaspargase before and after exposure to the freezing and thawing stress are shown in 

. The chromatographic profile shows that the peak corresponding to pegaspargase appears as a single sharp peak when not exposed to freezing and thawing stress; however, upon exposure to freeze-thawing, the peak appears as a split peak with an increase in a shoulder peak and a decrease in the principal peak corresponding to the pegaspargase. 

	Results obtained with SDS-PAGE analysis of the pegaspargase samples before and after exposure to the freezing and thawing stress are shown in 

. Qualitative analysis of pegaspargase samples by SDS-PAGE under non-reducing conditions shows no significant change in the polypeptide profile of the pegaspargase upon exposure to freezing and thawing stress.

	Chromatographic profiles obtained with samples of pegaspargase analyzed by HP-SEC followed by RI detection are shown in 

. The chromatogram shows no increase in the level of free PEG in the pegaspargase samples exposed to freeze-thaw stress.

Effect of high temperature on stability of pegaspargase. 

Pegaspargase formulated at about 8.8 mg/mL concentration in 49-mM sodium phosphate buffer in presence of 146-mM sodium chloride was exposed to high temperature conditions (40 °C ± 5 °C; 75% ± 5% RH) for a maximum up to one month as described in the previous section (Materials and methods). Samples were withdrawn at different time intervals and analyzed by HP-SEC and SDS-PAGE. 

	Chromatographic profiles obtained with samples of pegaspargase withdrawn at different time intervals are shown in 

. It is observed that the retention time and shape of the peak corresponding to pegaspargase changes with time of exposure at high temperature. The peak corresponding to pegaspargase shifts toward higher retention time with increase in exposure time at high temperature conditions. The deformation of the peak corresponding to pegaspargase also increases with time when exposed to high temperature. Results obtained with SDS-PAGE analysis of the pegaspargase samples withdrawn at different time intervals after exposure to high temperature over the period of one month are shown in 

. Qualitative analysis of pegaspargase samples by SDS-PAGE under non-reducing conditions show an increase in low molecular-weight species and the presence of free PEG as analyzed by two different staining methods using Coomassie R250 dye and barium iodide, respectively.

	In separate set of experiments, pegaspargase samples were exposed to high temperature conditions (i.e., 40 °C ± 5 °C; 75% ± 5% RH and 25 °C ± 5 °C; 60 % ± 5 % RH) for up to 15 days and 30 days, respectively. Samples were withdrawn at different time intervals and analyzed by HP-SEC followed by detection by an RI detector to check the effect of high temperature on release of PEG from the PEG-protein conjugate. Chromatographic profiles obtained with samples of pegaspargase exposed to high temperature conditions and withdrawn at different time intervals are shown in 

. It is observed that the level of free PEG increases with increase in exposure time under high-temperature conditions. 

	When exposed to higher temperature, pegaspargase shows changes in the retention time and shape of the peak corresponding to the pegaspargase protein as analyzed by HP-SEC. The deformation of the principal peak corresponding to the pegaspargase protein indicates a loss of structural integrity, and the shift toward the higher retention time suggests fragmentation of the protein molecule over the period of time upon exposure to the high-temperature conditions. The results obtained with SDS-PAGE analysis also show generation of low molecular weight species or fragmentation upon exposure to high-temperature conditions and corroborate the observations made through analysis of samples by HP-SEC. 

	Similarly, the significant increase in the shoulder peak and decrease in the peak corresponding to the pegaspargase probably indicates conformational changes in the pegaspargase protein when exposed to freeze-thaw stress. The SDS-PAGE analysis of pegaspargase protein exposed to freeze-thaw stress does not indicate any fragmentation or generation of low molecular weight species upon exposure to freeze-thaw stress. 

	The increase in the peak corresponding to the free PEG in the chromatograms obtained through analysis of the pegaspargase samples using HP-SEC followed by RI detection indicates removal of PEG from the protein backbone as a function of time when stored at high temperature conditions. When exposed to freeze-thaw stress, pegaspargase shows no increase in the peak corresponding to free PEG indicating no dissociation of the PEG from the protein backbone due to freezing and thawing. These results indicate that pegaspargase degrades through depegylation along with loss of structural integrity when exposed to high temperature; however, under freezing and thawing stress it does not show any depegylation. Under freezing and thawing conditions, the changes in the peak profile for the peak corresponding to pegaspargase were observed to be different than the changes observed when pegaspargase was exposed to high temperature stress, which indicates different effects on the structural properties of pegaspargase under each stress condition. 

	Oncaspar (PEG-asparaginase, Enzon Pharmaceuticals) in the form of liquid solution is known to have a short shelf-life (eight months) due to removal of PEG from the protein backbone. Also, the storage condition for pegaspargase drug substance is between +2 °C and +8 °C, unlike the majority of biological products that are generally stored in frozen form and are known to have longer shelf-lives of approximately two years. These storage limitations necessitate continuous or immediate conversion of drug substance material to the drug product material. Storage of drug substance material longer then the stipulated time frame between +2 °C and +8 °C reduces the shelf-life available to the final drug product as it is susceptible to depegylation. Unlike other biological products, it is not possible to store the pegaspargase drug substance material under frozen conditions without presence of any cryoprotectant, as freezing and thawing may lead to structural changes in the protein of interest and impact the biological activity or storage stability before it is converted to drug product. Operations such as shipment to distribution centers and pharmacies after manufacturing the drug product also take considerable time before the product gets delivered to the doctors for the treatment. Therefore, it becomes important to have maximum shelf-life available to the drug product material. 

	To bring flexibility in the production of the drug product, it is important to manufacture a drug substance that can be stored for a longer period of time so that the drug product can be manufactured based on the market demand and supply chain efficiency. Also, sudden excursions during shipping of the drug product material may affect the stability of the pegylated L-asparaginase protein. Product may be exposed to lower or higher temperature from the real-time storage conditions during shipment. These can lead to degradation of the pegylated L-asparaginase before it gets delivered to the patient and can show sub-optimal effect during treatment. 

	It is known that pegylation of L-asparaginase increases retention time of L-asparaginase in blood circulation and thereby increases the half-life. However, exposure of pegaspargase protein to both freeze-thaw and high-temperature stress indicated loss of protein integrity, probably due to significant alteration in the structural properties of the protein molecule. The freeze-thaw stress does not lead to any significant removal of PEG from the protein backbone; however, exposure to high temperature leads to depegylation. These observations indicate that the pegaspargase (pegylated L-asparaginase) follows different degradation pathways under different stressed conditions. Further, stabilization of pegaspargase in solution is required to avoid degradation of the molecule during manufacturing process, upon storage, and shipping. Stabilization against freeze-thaw can also help to decouple the manufacturing process of the drug product from that of the drug substance and can bring flexibility for manufacturing of drug product in a multi-product manufacturing facility.

	The authors are thankful to Dr. Sanjeev Kumar Mendiratta, president, and Mr. Chandresh Bhatt, research associate, both in the department of Biotechnology at the Zydus Research Centre, Cadila Healthcare, Ahemdabad, for their scientific guidance and technical support.

	15. Shmuel Zalipsky, Enzon Pharmaceuticals Inc, “Active carbonates of polyalkylene oxides for modification of polypeptides,” US patent 5612460A, Apr. 1989.


	Vol. 43, No. 4
	April 2019
	Pages: 40–48

	When referring to this article, please cite it as C. Patel, S. Bandyopadhyay, and G. Patel, "Degradation Pathways: A Case Study with Pegylated L-Asparaginase," 

, is vice-president, both in the Department of Biotechnology, Zydus Research Centre, Cadila Healthcare Ltd., Ahmedabad, Gujarat, India; 

, is associate professor in the Department of Pharmaceutics & Pharmaceutical Technology, Ramanbhai Patel College of Pharmacy at the Charotar University of Science and Technology, Changa Campus, Changa- 388 421, Anand, Gujarat, India, gayatripatel.ph@charusat.ac.in, +91 2697 265162.

To whom all correspondence should be addressed.

Articles

It is important to address manufacturing problems associated with the shorter shelf-life of pegylated L-asparaginase (pegaspargase) upon long-term storage in the form of a solution. 