Abstract
Recently, new and advanced ideas have been presented on the value of polymer-based syringes for improved safety, better strength, reduced aggregation, and the prevention of drug degradation. In this report, our findings on drug degradation from protein oxidation will be presented and discussed. Commonly, dissolved oxygen is one of the factors for causing protein degradation. Due to the nature of higher gas permeability in polymer-based syringes, it was thought to be difficult to control the oxygen level during storage. However, this report demonstrates the appropriateness of combining the use of an oxygen absorber within the secondary packaging as a deoxygenated packaging system. In addition, this report suggests that another factor to enhance protein oxidization is related to radicals on the syringe barrel from sterilization by irradiation. We demonstrate that steam sterilization can minimize protein oxidization, as the protein filled in steam sterilized syringe is much more stable. In conclusion, the main oxidation pathway of a protein has been identified as dissolved oxygen and radical generation within a polymer container. Possible solutions are herewith presented for controlling oxidation by means of applying a deoxygenated packaging system as well as utilizing steam sterilization as a method of sterilization for prefillable polymer syringes.
LAY ABSTRACT: There have been many presentations and discussions about the risks associated with glass prefilled syringes. Advanced ideas are being presented on the value of polymer-based syringes for improved safety, better strength, reduced protein aggregation, and the prevention of drug degradation. Drug degradation based on protein oxidation is discussed in this report. Identification of the main factors causing this degradation and possible solutions available by using polymer-based syringes will be presented. The causes of protein oxidation have been identified as dissolved oxygen and radicals generated by the applied method of sterilization. The oxidation reaction created by dissolved oxygen within the drug product can be effectively inhibited by controlling the removal of the oxygen through the use of a deoxygenated packaging system. This packaging system can control the level or complete removal of oxygen from the primary container and the secondary packaging system. Protein oxidation induced by the formation of radicals from sterilization by irradiation is another critical aspect where it was thought that various sterilization methods were acceptable without loosing drug product quality. However, this report is first to demonstrate that gamma sterilized polymer-based syringes accelerated protein oxidation by radical generation; this effect can be prevented by means of steam sterilization.
Introduction
Around the world, prefilled syringes (PFSs) are preferred as a parenteral drug container to bring various advantages such as ease of use, minimizing errors in clinical use, and the ability for self-injection. The applications for and interest in PFSs are increasingly relevant due to the heightened importance of biopharmaceuticals in parenteral drug development. In comparing the use of polymer PFSs as a primary container for Japan versus the global market, an interesting trend is observed in which most of PFSs in the Japanese market are made from polymers whereas glass is still predominantly used outside of Japan.
Thus the interest in the field of parenteral packaging within the pharmaceutical industry is moving towards polymer-based PFS systems, although glass PFSs are still preferred in the global market at present (1). The reason for this is due to an increase in the number of recalls of pharmaceutical drug products by the U.S. Food and Drug Administration (FDA) (2). Main reasons for recalls are issues related to particulate matter and the contamination by glass particles, cracks, breakage, and delamination (2). When protein drug products are degraded due to aggregation with foreign substances, the toxicology profile is altered and the resulting product may become harmful to the patient (3⇓⇓⇓⇓–8). For instance, recently it has been reported that silicone oil can induce protein aggregation, and such aggregation may lead to unexpected immunogenicity issues (9, 10). Terumo has developed a silicone/oil–free (SOF) PFS system comprised of a polymer-based syringe barrel and i-coating™ stopper. i-coating™ technology is a proprietary process of bonding a blended silicone resin to the stopper substrate for improved break-loose and gliding forces between the barrel and stopper (11). Using this SOF system reduces the risk of protein aggregation induced by silicone oil and provides consistent performance of the break-loose and glide forces when the product is stored for an extended time, thereby providing a repeatable and reliable performance not found in siliconized glass systems.
Protein degradation due to oxidation is well known for small- and large-molecule drug products. Dissolved oxygen occurs naturally in the drug product during the formulation and filling processes. Nitrogen blanketing and other techniques are often used to reduce the effect of oxygen on the product. This article will discuss how using a system approach to design a deoxygenating package can prevent degradation due to the presence of oxygen. In addition, this article will also discuss how free radicals created during sterilization can further contribute to oxidation and degradation of drug products stored in a container.
The control of dissolved oxygen to prevent oxidation can be performed by several methods, including adding antioxidizing agents to the drug formulation and/or controlling the residual oxygen within the primary container during storage. Glass PFSs were found to be less effective when used in a deoxygenated packaging system when compared to polymer PFSs. Terumo has experience in the Japanese PFS market to control oxygen concentration in a drug solution by using deoxygenated packaging systems and has applied this technology to control dissolved oxygen to amounts close to zero. As such, we have already launched drug products successfully within the Japanese market, such as multivitamins and epinephrine for injection. With our deoxygenated packaging system, we were able to control oxygen content within the drug product during storage. In this report, the elimination profile of the dissolved oxygen in PFSs and the effect of the deoxygenated packaging system on drug stability are presented.
Another key factor for drug oxidation is the formation of radicals as described above. Generally, it is well known that radiation enhances the oxidation of a drug because radiation exposure generates free radicals and these radicals act as an oxidation enhancer, resulting in poor stability of the drug product (7, 8). In this article we will present additional information on how the method of sterilization of a PFS system can affect the drug quality when the drug is filled into a PFS sterilized with high-energy sterilization methods such as gamma radiation.
Materials and Methods
Materials
A PLAJEX™ PFS [1 mL staked needle (27G)] SOF system was provided by Terumo Co. (Tokyo, Japan). PLAJEX™ is a PFS system with a polymer barrel made of cyclo olefin polymer (COP) and a butyl rubber plunger stopper coated by use of the i-coating™ technology (a proprietary SOF coating). Erythropoietin (EPO) and Glu-C were purchased from Sigma-Aldrich (St. Louis, MO). All buffer salts (sodium phosphate monobasic, sodium phosphate dibasic, and sodium chloride) were purchased from Kanto Chemical Co. (Tokyo, Japan). All other chemicals used in this study were analytical-grade.
Preparation of Sterilized Polymer-based PFSs
The polymer-based PFSs were sterilized by steam and gamma irradiation. The intensity in gamma sterilization was conducted at 25 kGy for protein stability testing. PFS barrels sterilized by gamma irradiation at 25 and 50 kGly were used for the Fourier transform infrared (FTIR) study. Steam sterilization was conducted under conditions of 121 °C and 20 min.
Measurement of Dissolved Oxygen
Water was filled into the PFS system, and then left at ambient conditions. At a predetermined time, the dissolved oxygen level was measured with an oxygen meter (OXY-4 micro, PreSens Precision sensing GmbH, Regensberg, Germany). The objective of this study was to clarify how effective the deoxygenated package system was in controlling the dissolved oxygen. Two groups were made: non-packaged samples and deoxygenated packages. For the deoxygenated package group, we developed the packaging system composed of a PFS, a blister package with a sealed cover, and an oxygen absorber as shown in Figure 1. To prevent gas permeation through the secondary packaging materials, we adopted a blister film with a three-layer structure having low gas-permeable properties.
Deoxygenated packaging system designated for oxygen control.
Protein Oxidation Study
EPO was dissolved with aqueous solution consisting of 2 mM Na2HPO4 and 0.06 mg/mL polysorbate 80 to give a concentration 24,000 IU/mL. After complete dissolution, the PFSs sterilized by steam or gamma irradiation were filled with 1.0 mL of EPO solution and stoppered with plunger stoppers leaving a predetermined head space of 0.2 mL. The filled PFSs as prepared by the above procedure were stored at 25 °C and 65% relative humidity (RH) for 2 and 3 months. Thereafter the drug product was evaluated by high-performance liquid chromatography (HPLC) as described below.
Analytical Method: Oxidized Methionine in EPO
Analysis of oxidized methionine in EPO was conducted with HPLC using a modified method as referenced in Ohta et al. (12). Briefly, a mixture of 100 μL of protein solution and 400 μL ammonium acetate solution with 100 mM and pH 8.0 was placed into a Amicon Ultra-0.5 10 K (Millipore Ireland Ltd., County Cork, Ireland) recipient, and was centrifugated at 14,000 g for 15 min. The unfiltrated sample remaining on the filter was collected carefully. To collected sample, 100 mM ammonium acetate solution pH 8.0 was added to make 50 μL solution as a final volume. To separate the oxidized methionine (Met-Oxy) and intact methionine (Met) fragment, 1 μg/mL Glu-C in 100 mM ammonium acetate pH 5.6 was added to the samples. Thereafter the samples were incubated for 37 °C for 24 h. Finally, the obtained sample was diluted with pH 8.0, 100 mM ammonium acetate solution pH 8.0, and then the HPLC analysis (Shimadzu, Kyoto, Japan) was conducted.
The HPLC condition was as follows:
Mobile phase A: 0.05% trifluoro acetic aqueous solution
Mobile phase B: acetonitrile containing 0.05% trifluoro acetatic acid
Column: Inertsil ODS-3 (5 μm, 250 mm x 2.1 mm i.d.)
Column temperature: 40 °C
Injection volume: 30 μL
Total flow rate: 0.25 mL/min
Wavelength: 280 nm
Flow mode: linear gradient mode
Time program
In Figure 2, HPLC chromatogram obtained following the above method are shown, and according to the following equation the percent of Met-Oxy was calculated.
Chromatogram of (A) non-treated EPO, (B) Glu-C digested EPO, and (C) Glu-C digested sample after treating EPO with perchloric acid/hydrogen peroxide. Sample preparation for (C) was conducted as follows: After mixing EPO and 0.7% perchloric acid with a mixture ratio of 10:4, hydrogen peroxide was added to give 0.0002% (v/v) as a final concentration. Finally, this solution was incubated at 37 °C for 30 min to transform Met to Met-Oxy.
FTIR Spectroscopy
The PFS barrels sterilized by steam and gamma irradiation at 25 and 50 kGy were cut into square specimens of 3 × 3 mm. These specimens were analyzed using a FTIR spectrometer (Spectrum100, PerkinElmer, Wellesley, MA) equipped with a universal attenuated total reflectance (ATR) sampling accessory. ATR-FTIR spectra were recorded at a resolution of 4 cm−1, and 16 scan per specimen were averaged in a wave number range from 650 to 4000 cm−1.
Results and Discussion
Dissolved Oxygen Control with Deoxygenated Packaging System
Dissolved oxygen was one of the factors causing protein degradation through the oxidation pathway. For preventing the oxidation of product, strategies have to be taken such as adding certain excipients into the drug formulation for controlling the oxygen level during manufacturing.
Another method by example are the Terumo products of epinephrine for injection and multivitamins for infusion packaged into a deoxygenated packaging system composed of low gas-permeable foil and the inclusion of an oxygen absorber as shown in Figure 1.
Under this packaging system, the dissolved oxygen level was dramatically decreased in the solution within the PFS system as compared to the non-deoxygenated PFS system (Figure 3). This decrease in dissolved oxygen level within the glass PFS irrespective of the deoxygenated packaging system was not observed (data not shown). The differences of oxygen elimination between glass and this polymer-based PFS are considered to be due to the difference in the gas permeability characteristics because commonly the gas permeability is extremely low for glass PFSs compared with the polymer-based PFSs (13). In other words, only polymer-based PFSs can achieve oxygen control with the package system while maintaining adequate container closure.
Elimination profile of dissolved oxygen under deoxygenated packaging system. ○: without deoxygenated packaging system, •: with deoxygenated packaging system. Data represent the mean ± standard deviation (SD) (n = 3). Detailed packaging structure is depicted in Figure 1.
Theoretically, the dissolved oxygen level within a deoxygenated packaging system can be controlled to a predetermined level between the blister package and the headspace of the PFS. Any oxygen left in the system will equilibrate between all areas controlled by the system. This type of oxygen control cannot be found using traditional purging and blanketing techniques during filling. From these points, the combination of a polymer-based PFS system and a deoxygenated packaging system as described here is the easiest and most effective method for controlling the dissolved oxygen level.
To show the effectiveness of the proposed deoxygenated packaging system, a comparative study was conducted to reveal the difference in the protein oxidation between the product packaged with or without the deoxygenated packaging system. In this study, EPO aqueous solution was used as model protein solution, which underwent the oxidation in the methionine part in EPO (Met-Oxy) (7). The PFS barrels sterilized by steam were filled with EPO solution as prepared above, and the PFSs were placed under 25 °C and 65 % RH conditions for 3 and 6 months.
As shown in Figure 4, the drug product without the deoxygenated packaging system revealed a significant higher Met-Oxy ratio than the samples kept within the deoxygenated packaging system at all time points except time zero. From the abovementioned findings, our concept has been proven: the deoxygenated packaging system for polymer-based PFSs is very useful for drug products that are vulnerable to degradation by dissolved oxygen.
Comparison of protein stability with and without deoxygenated packaging system. ○: without deoxygenated packaging system. •: with deoxygenated packaging system. The syringes were stored at 25 °C and 65% RH. Data represented the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 against data of “with-deoxygenated” packaging system.
Effect of the Sterilization Method on Protein Stability and Prediction of Radical Generation within a Polymer PFS
Decomposition of a biopharmaceutical drug product may result in unexpected side effects and/or reduced effectiveness, therefore controlling and preserving the product quality is of paramount importance. Polymer-based PFS barrels can be sterilized by ethylene oxide gas, steam, and irradiation. It is commonly understood that radicals are generated by irradiation, but so far there are no reports that show the impact of radical formation on protein stability.
Sterile, single-use PFSs (often called disposable PFSs) are commonly sterilized by gamma or electron-beam sterilization. There are no large risks from the effect of radicals on drug stability because the drug solution taken into these PFSs have a relative short contact period within the PFS and are immediately administrated to patients. However, in cases of PFS systems intended as a primary container for medicinal products, the storage period is substantially longer and the generated radicals may have a greater impact on the drug product quality. To clarify the influence on protein stability after filling into PFSs sterilized by gamma radiation at 25 kGy or by steam, a comparative study was conducted. The model protein used EPO as well investigated the deoxygenated package system.
As shown in Figure 5, the percentage of Met-Oxy was dramatically different as a result of the applied method of sterilization. In case of steam-sterilized PFSs, the percentage of Met-Oxy did not change over time. On the other hand, in case of gamma irradiation sterilization, the percentage of Met-Oxy was increased over time and the value of this ratio reached levels of about 30% after 12 weeks of storage. For steam-sterilized PFSs, the percentage of Met-Oxy was only 3% at the same time point. In general, it is assumed that a protein oxidation is due to the dissolved oxygen or free radicals generated by irradiation. In this study, the dissolved oxygen level is the same for PFSs sterilized by steam and gamma irradiation because the sample preparation procedure including drug preparation, filling volume, and air headspace into the PFSs was completely the same. Therefore, it is thought that the observed difference in protein oxidation is related to the amount of radical generation between steam sterilization and sterilization by gamma irradiation. A materials analysis was conducted at the surface of PFS barrels sterilized by steam and gamma irradiation to demonstrate the different effects of these sterilization techniques.
Comparison of Met-Oxy profile after filling into syringes sterilized by (•) gamma irradiation sterilization and (○) steam sterilization. The syringes were stored at 25 °C and 65% RH. Data represent the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 against data of steam sterilization.
The results of FTIR analysis are shown in Figure 6; the different IR spectra were confirmed among steam-sterilized and gamma irradiation–sterilized PFSs. For steam-sterilized PFSs, the IR spectrum was not changed. On the other hand, in the case of gamma irradiation–sterilized PFSs, the IR spectrum was changed approximately 1730 cm−1, which was derived from the C=O bond. This result suggested that the PFS barrel was oxidized by gamma irradiation sterilization, and we speculated that the oxidation of the PFS barrel is due to the generated radicals. Further investigation is needed to confirm that the generated radicals caused the protein destabilization through oxidation.
FTIR spectra of non-sterilized, steam-sterilized and gamma irradiation–sterilized syringe barrels. The peak at around 1730cm−1 (surrounded with dashed line) corresponds to C=O.
Conclusion
The root causes of protein oxidation were identified as being dissolved oxygen within the drug product and the effect from radicals generated within the container by irradiation sterilization. Oxidation reaction due to dissolved oxygen can be avoided by controlling the oxygen content by reduction or complete removal of the dissolved oxygen using a deoxygenated packaging system. Radical generation within a container due to irradiation is not well known and is presented here for discussion. This presents a very important and critical aspect as compared to the control of dissolved oxygen because it was thought that any sterilization method for prefillable primary containers was acceptable and would not result in reduction of product quality.
Through our experiments and results, the main oxidation pathway of a protein has been identified as dissolved oxygen and radical generation within a polymer container. This report also demonstrates several solutions for controlling oxidation by means of applying a deoxygenated packaging system as well as utilizing steam sterilization as a method of sterilization for polymer PFSs.
Conflict of Interest Declaration
The authors declare that they do not have any financial or non-financial competing interests related to the content of the manuscript.
- © PDA, Inc. 2015
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