Gayatri Patel is associate professor of pharmaceutics and pharmaceutical technology at Charotar University of Science and Technology, Changa, Gujarat, India

Gayatri Patel

	Submitted: Submitted: May 1, 2019, Accepted: June 14, 2019

Depending on the polyethylene glycol reagent and chemistry used to manufacture them, pegylated products may exhibit different degradation patterns under different storage conditions. To ensure product quality, it is essential to prevent degradation, which can occur when temperatures exceed optimal levels during manufacturing, storage, and shipping. To get a better understanding of degradation patterns, research was done to evaluate the stability of pegylated products under various storage conditions.

	This article discusses work that was done to examine the effect of repeated freezing and thawing cycles on the stability of peginterferon alfa-2b, and to study its storage stability in frozen as well as liquid form. It also discusses how results might pertain to various other pegylated products. Size-exclusion chromatography, circular dichroism, and fluorescent spectroscopy were all used to evaluate stability under different conditions. Results suggest that storage in liquid form can lead to degradation at temperatures between +2 °C and +8 °C.

	Interferons exhibit both antiviral and antineoplastic effects (1). Interferon alfa-2b (Intron A), interferon alfa-2a (Roferon-A), and interferon beta-1b (BETAFERON) are approved for various indications either alone or combined with other agents. Both alfa-2b and alfa-2a types have half-lives of less than 12 hours, necessitating multiple injections (at least three times per week) for the duration of treatment, which can range from three weeks to 24 months (2,3).

	Longer-acting versions of both alfa 2b and alfa 2a have been developed to help offset this problem. Examples include peginterferon alfa-2b (Pegintron/ViraferonPeg) and peginterferon alfa-2a (Pegasys). Pegintron/ViraferonPeg has been approved as part of a combination regimen to treat chronic hepatitis C (CHC) in patients with compensated liver disease. Pegasys is indicated for CHC and chronic hepatitis B.

	Most of the first generation of pegylated products (i.e., Adagen (Pegademase), Oncaspar (Pegaspargase), Pegintron (peginterferon alfa-2b) and Pegasys (peginterferon alfa-2b) were developed with commercially available acylating PEG reagents (4). Adagen and Oncaspar were prepared using monomethoxy PEG activated with succinimidyl succinate (mPEG-SS) (5). Pegintron is prepared by conjugating interferon alfa-2b with a single chain 12 kDa PEG activated with succinimidyl carbonate (mPEG-SC), while Pegasys is prepared by conjugating interferon alfa-2a with N-hydroxysuccinimide (NHS) activated 40 kDa branched PEG molecule (6,7). Pegintron is a mixture of biologically active mono-pegylated positional isomers so that most of the pegylation occurs at the Histidine 34 (His34) residue. Within this residue, a urethane-like bond that is formed by succinimidyl carbonate (SC) conjugation chemistry with the imidazole ring of His34 is hydrolytically labile. In contrast, however, Pegasys is produced utilizing N-Hydroxysuccinimide (NHS) chemistry through amide bond formation. These bonds are not susceptible to spontaneous hydrolysis (8).

	For any pegylated interferon, changes in protein temperature stability have been found to depend on the coupling chemistry, degree of pegylation, number of protein subunits, and formulation involved. It is known, for example, that pegylation has no effect on the secondary or tertiary structure of interferon alfa-2b and interferon alfa-2a. Not only the conformational stability of the protein molecule but the stability of the PEG-protein conjugate is important if the drug is to exhibit the desired biological activity and bioavailability. For example, Oncaspar (Pegaspargase), which is produced by conjugating mPEG-succinimidyl succinate (SS) with L-asparaginase, has a short shelf-life when supplied as a liquid solution, where the enzyme activity of L-asparaginase increases upon depegylation (9). In addition, pegaspargase shows different degradation pathways when exposed to high temperature and freeze-thawing stress (10). Pegylated products produced using different PEG reagents and pegylation chemistries (i.e., pegylation with mPEG-SS vs. mPEG-SC) may follow different degradation pathways under different storage conditions. Product may be exposed to sudden temperature excursions during the manufacturing process, storage, and shipping, which can affect product stability. Stability of pegylated products should be evaluated under various storage conditions, considering the nature of the product. The authors researched the effect of repeated freezing and thawing on the stability of peginterferon alfa-2b, as well as the stability of frozen peginterferon alfa-2b and liquid material maintained between +2 °C and +8 °C. Research focused on identifying the degradation pattern of peginterferon alfa-2b under these storage conditions, and were discussed with respect to the stability of various pegylated products.

. Interferon alfa-2b was produced in-house using recombinant DNA technology and pegylated using activated polyethylene glycol with a particle diameter of 12 kDa. Peginterferon alfa-2b was produced by pegylating interferon alfa-2b with 12-kDa monomethoxy polyethylene glycol succinimidyl carbonate (mPEG-SC), to the innovator product, PEGINTRON. PEG conjugation was carried out through integration of a carbamate (urethane) linkage, between N-atoms of the imidazole side-chain of His34 or the μ-NH2 group of N-terminal Cysteine residue, or the Ïµ-NH2 group of Lysine side-chains of interferon alfa-2b and a 12-kDa mPEG-SC molecule.

	Pegylation was carried out at pH 6.5 in the presence of a molar-excess amount of mPEG-SC over the protein amount. After pegylation, multiple-column chromatography (using GE’s Amersham Biosciences’ AKTA) was used to purify the peginterferon alfa-2b, mainly in monopegylated form.

	After chromatography, peginterferon alfa-2b was brought into 10 mM sodium succinate buffer using succinic acid and sodium hydroxide (Merck) and maintained at a pH of 6.8. A 0.2-µm sterile filter (Sartopore 2, Sartorius Stedim Biotech GmbH) was then used to filter the protein solution.

	Degradation of frozen vs. liquid peginterferon alfa-2b

	Peginterferon alfa-2b’s degradation pattern was assessed by analyzing results of repeated freezing and thawing cycles on the material. To check the effect of repeated freezing and thawing, peginterferon alfa-2b solution was aliquoted with 1 mL solution in a 10-mL container (Thermo Scientific) made of polytetrafluorethylene (PTFE) (Teflon, DuPont). Samples were frozen at or below –70 °C in the deep freezer (Thermo Electron Corporation, Model No.: ULT1740-3-V40). Thawing was done at room-temperature and was considered complete when the frozen mass had been completely converted into liquid. To check the degradation of liquid peginterferon alfa-2b protein, liquid samples were stored between +2 °C and +8 °C in the container, while samples that had been frozen at or below -70 °C were used to determine the stability of frozen material. Samples were withdrawn at different time intervals and analyzed by different test parameters.

	The presence of free interferon alfa-2b was assumed to indicate that depegylation had occurred in the test samples. High-performance size-exclusion chromatography (HP-SE), utilizing an ultraviolet (UV) detector, was used to measure samples to determine stability. Measurements were performed on a Shimadzu LC 2010-CHT series HPLC system equipped with a Tosoh Biosciences G3000SWXL column using a TSK gel (7.8-mm ID × 30.0 cm/L). Before injecting the sample, the column was pre-equilibrated with 0.2 M phosphate buffer containing 10% ethanol at a pH of 6.8 and a flow rate of 0.5 mL/min at an oven temperature of 25 °C. After the column was equilibrated, 10-µg samples were injected and analyzed in isocratic mode at a flow rate of 0.5 mL/min. Chromatographic separation was monitored at 214 nm utilizing the UV detector.

	Test samples exposed to repeated freezing and thawing cycles were also tested to determine the structural integrity of peginterferon alfa-2b protein under these conditions by comparing them with initial sample that had not undergone freezing or thawing. Far-UV circular dichroism (CD) spectroscopy and spectrofluorometry (using a Jasco J-1500 instrument equipped with an MCB-100 Peltier-based temperature-controlled assembly) was used to analyze the secondary structure of peginterferon alfa-2b samples.

	A smoothing algorithm was used on the CD spectrum for baseline correction, and peak position was identified by using Spectra Manager software. Fluorescent spectroscopy (using a JASCO FP-8300 instrument equipped with a MCB-100 Peltier-based temperature-controlled assembly) was then used to evaluate the tertiary structure of peginterferon alfa-2b protein in the test samples. The temperature of the sample holder was controlled, and samples were incubated at 20 °C for 5 min under stirring conditions. Samples were then excited at 280 nm to capture total intrinsic fluorescence emission. Emission spectra were collected in the range of 300–450 nm. Again, a smoothing algorithm was used for smoothing and baseline correction, and Spectra Manager software was used to identify the maximum peak position.

Effects of repeated freeze-and-thaw cycles on the stability of Peginterferon alfa 2-B

. Peginterferon alfa-2b protein present in 10-mM sodium succinate buffer at a pH of 6.8 was exposed to freeze-thaw stress as described previously. Samples were exposed to three consecutive freezing and thawing cycles and evaluated to determine whether there had been an increase in the level of free interferon alfa-2b by HP-SEC. 

 shows the chromatographic profiles obtained with the samples of peginterferon alfa-2b before and after exposure to the freezing and thawing stress. The chromatogram obtained for the test samples exposed to freezing and thawing cycle shows no increase in the peak corresponding to free interferon alfa-2b, indicating no further increase in the level of free interferon alfa-2b upon repeated freezing and thawing compared to the result obtained with the unexposed initial sample.

	Secondary structure analysis of the test samples of peginterferon alfa-2b exposed to repeated freezing and thawing was conducted by CD spectroscopy in the far-UV (260–190 nm) region to assess the effect on structural integrity of protein. As illustrated in 

, CD spectra obtained with the test samples exposed to repeated freezing, and thawing cycles were observed to overlap with the spectra obtained for the unexposed initial sample in the far-UV region. The overlapping CD spectra obtained with all the samples showed absorbance minima at wavelengths 208 nm and 222 nm, typical for cytokine molecules, which remain unaltered upon repeated freezing and thawing of the peginterferon alfa-2b in presence of 10 mM sodium succinate buffer at pH 6.8.

 shows results obtained with peginterferon alfa-2b test samples analyzed by spectrofluorometry to evaluate the effect of repeated freezing and thawing on tertiary structure. The fluorescence spectra obtained with the test samples that had been exposed to repeated freezing and thawing were observed to overlap with spectra obtained for unexposed initial samples, indicating no effect on tertiary structure of peginterferon alfa-2b protein.

	Stability of peginterferon alfa-2b: frozen vs. liquid form

	Peginterferon alfa-2b in 10-mM sodium succinate buffer at pH 6.8 was stored in liquid form between +2 °C and +8 °C, and in frozen form at temperatures at or below –70 °C for three months to check for any increase in the level of free interferon alfa-2b under both the storage conditions.

	Samples were withdrawn at different time intervals and analyzed by HP-SEC. The level of free interferon alfa-2b in the test samples withdrawn at different time intervals from both the storage conditions is shown in 

. When stored as liquid solution between +2 °C and +8 °C, the level of free interferon alfa-2b increased, with time indicating depegylation under these storage conditions. However, when stored under frozen conditions, even for up to three months, the material showed no significant change in the level of free interferon alfa-2b observed. In a separate study, stability of peginterferon alfa-2b was established at least up to 12 months when stored at or below –70 °C without any significant depegylation.

	Experimental results demonstrate that peginterferon alfa-2b protein can withstand up to three multiple freeze-thawing cycles without showing any loss of structural integrity and depegylation. When stored under frozen conditions (i.e., at or below –70 °C), the material does not show any increase in level of free interferon alfa-2b for at least three months, showing that no depegylation had occurred. These results stand in contrast to what was observed with the liquid form.

	For most biological products, drug substance is generally stored in the frozen form and known to have longer shelf-life of at least about two years before it gets converted into the drug product upon formulation. However, it is known that the manufacturing process of Oncaspar liquid drug product (Enzon Pharmaceuticals Inc.), involves continuous processing of the drug substance to produce drug product wherein the hold time stability of the drug substance material is established between +2 °C and +8 °C.

	In a separate study, authors have found that pegaspargase does not show stability upon freezing and thawing (10). Unlike results obtained with pegaspargase, which is a multi-subunit protein highly susceptible to conformational changes upon freezing and thawing, peginterferon alfa-2b produced using mPEG-SC with carbamate linkage showed high stability when exposed to repeated freezing and thawing. These observations suggest that different protein molecules pegylated with different PEG reagents and using different pegylation chemistries may exhibit different degradation patterns when exposed to freezing and thawing stress. Therefore, storage conditions during various manufacturing process steps must be selected very carefully, based on the pegylation chemistry utilized, the type of protein molecule involved (i.e., single subunit vs. multiple subunits) and degree of pegylation.

	Oncaspar’s susceptibility to conformational changes of the protein molecule upon freeze-thawing and depegylation when stored at +2 °C and +8 °C suggests that the drug substance material should be immediately and continuously converted to drug product during fill/finish manufacturing so that the material does not have to be stored.

	Based on results obtained with the peginterferon alfa-2b samples stored at or below –70 °C, peginterferon alfa-2b can be stored in frozen form. This allows drug substance and drug product manufacturing to be decoupled, removing the need for continuous processing. The storage stability of peginterferon alfa-2b in frozen form can enhance manufacturing flexibility, particularly for production campaigns in a multi-product facility.

	Another pegylated product, Pegasys (peginterferon alfa-2a) which is prepared by conjugating interferon alfa-2a with an NHS-activated, 40-kDa branched PEG molecule with stable amide linkage supplied as a liquid solution with the shelf-life of two years when stored between +2 °C and +8 °C whereas Oncaspar has a shelf-life of only eight months, due to removal of PEG from its protein backbone.

	Like Oncaspar, peginterferon alfa-2b remains unstable when stored as a liquid solution between +2 °C and +8 °C and degrades through depegylation as these experiments confirmed. Due to the unstable linkages present in Oncaspar and peginterferon alfa-2b, it is crucial to produce the drug product material as a lyophilized powder and reconstitute it just beforethe injection.

	Peginterferon alfa-2b exhibited stability after repeated freezing and thawing stress cycles, without the addition of any cryoprotectant. No impact was observed on the protein molecule conformation upon repeated freezing and thawing. When stored in liquid form, however, even at +2 °C to +8 °C, peginterferon alfa-2b shows significant depegylation. When stored in frozen form, it remains stable without any depegylation. Pegylated products may exhibit different storage stabilities when exposed to repeated freezing and thawing, based on the PEG reagent and pegylation chemistry utilized. It is thus very important to evaluate the storage stability of pegylated products at various steps of the manufacturing process. Identifying degradation patterns that will occur under different storage conditions can play important role in optimizing both drug substance and product manufacturing process designs.

	The authors thank Sanjeev Kumar Mendiratta, president, biologics R&D and manufacturing, and Chandresh Bhatt, research associate, biologics R&D, at Cadila Healthcare Ltd.’s Zydus Research Center in Ahmedabad for their guidance. 

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	Chintan Patel is researcher and Sanjay Bandyopadhyay is vice president, both at Cadila Healthcare Ltd.’s Zydus Research Center in Ahmedabad, Gujarat, India; Gayatri Patel* (gayatripatel.ph@charusat.ac.in), is associate professor of pharmaceutics and pharmaceutical technology at Charotar University of Science and Technology, Changa, Gujarat, India.

	*To whom all correspondence should be addressed.

Vol. 43, No. 10
	October 2019
	Pages: 34–42

	When referring to this article, please cite it as C. Patel, S. Bandyopadhyay, and G. Patel, “Optimizing Manufacturing Based on the Storage Stability of Pegylated Products,” 

Articles

Research into peginterferon alfa-2b’s degradation pathways suggest that drug substance be immediately and continuously converted to drug product when the material is in liquid form.