Chemical-gas Sterilization of External Surface of Polymer-based Prefilled Syringes and Its Effect on Stability of Model Therapeutic Protein.

To reduce the risk of infection during intravitreal injections, the external surface of prefilled syringes (PFSs) must be sterilized. Usually, ethylene oxide (EO) gas or vaporized hydrogen peroxide (VHP) is used for sterilization. More recently, nitrogen dioxide (NO2) gas sterilization has been developed. It is known that gas permeability is approximately zero into glass-PFSs. However, polymer-PFSs (P-PFSs) have relatively high gas permeability. Therefore, there are concerns about the potential impact of external surface sterilization on drug solutions in P-PFSs. In this study, P-PFSs [filled with water for injection (WFI) or human serum albumin (HSA) solution] were externally sterilized using EO, VHP, and NO2 gases. For the WFI-filled syringes, the concentration of each gas that ingressed into the WFI was measured. For the HSA solution-filled syringes, the physical and chemical degradation of HSA molecules by each sterilant gas was quantified. For the EO- or VHP-sterilized syringes, the ingressed EO or hydrogen peroxide (H2O2) molecules were detected in the filled WFI. Additionally, EO-adducted or oxidized HSA molecules were observed in the HSA-filled syringes. In contrast, the NO2-sterilized WFI-filled syringes exhibited essentially immeasurable ingressed NO2, and protein degradation was not detected in HSA-filled syringes.

Impact of Ethylene Oxide Sterilization of Polymer-Based Prefilled Syringes on Chemical Degradation of a Model Therapeutic Protein During Storage.

Materials from prefilled syringe systems-such as silicone oil, tungsten, glass, and rubber-may enhance therapeutic protein aggregation and particle formation. Also, the sterilization method used for syringes may impact aggregation and chemical degradation of biologics during storage. Syringes are generally sterilized by radiation, ethylene oxide gas (EO), or steam. Among the sterilization methods, EO has the potential to cause chemical degradation by the formation of adducts with susceptible amino acid residues in the protein. In this study, EO- and steam-sterilized syringes were compared to determine the influence of residual EO on human serum albumin (HSA) degradation. Although the amount of residual EO in the EO-sterilized syringes was less than 220 µg/syringe, well below the International Organization for Standardization limit, EO adduction to cysteine (Cys) and methionine (Met) in HSA was observed by liquid chromatography and mass spectrometry analysis. The EO adduct ratio of HSA stored for 2 weeks in EO-sterilized syringes was about 45%. In contrast, no chemical degradation was observed in HSA formulation stored in steam-sterilized syringes. Because of the propensity of EO to readily form adducts with proteins, an alternative to EO sterilization should be used for prefilled syringes that will be used for therapeutic protein products.

Effects of Product Handling Parameters on Particle Levels in a Commercial Factor VIII Product: Impacts and Mitigation.

To reduce the risk of immunogenicity that may be caused by therapeutic protein products, it is important to properly characterize subvisible particles and to develop strategies to reduce the levels of particles delivered to patients. In the present study, by using state-of-the-art methods to quantify particle levels, we found that the factor VIII product, Kogenate FS, contained relatively high levels of protein particles and silicone oil droplets, the vast majority of which were submicron in size. In a test of effects of product mishandling, the Kogenate FS vial was shaken instead of swirled during reconstitution. Levels of silicone oil droplets and protein particles were increased. In contrast, these levels were greatly reduced by 2 mitigation strategies tested, using a nonsiliconized syringe for the diluent container or using submicron pore size syringe filters during simulated infusion. Thus, to avoid potential adverse effects due to mishandling-induced increases in particle levels, it is important to educate end-users about proper product handling. Furthermore, effective particle mitigation and reduction strategies should be developed for factor VIII, and other therapeutic protein products. Such efforts could lead to clinically useful approaches to reduce the levels of particles delivered to patients and to an associated reduction in adverse immunogenicity.

Human case of bacteremia caused by Streptococcus canis sequence type 9 harboring the scm gene.

Streptococcus canis (Sc) is a zoonotic pathogen that is transferred mainly from companion animals to humans. One of the major virulence factors in Sc is the M-like protein encoded by the scm gene, which is involved in anti-phagocytic activities, as well as the recruitment of plasminogen to the bacterial surface in cooperation with enolase, and the consequent enhancement of bacterial transmigration and survival. This is the first reported human case of uncomplicated bacteremia following a dog bite, caused by Streptococcus canis harboring the scm gene. The similarity of the 16S rRNA from the infecting species to that of the Sc type strain, as well as the amplification of the species-specific cfg gene, encoding a co-hemolysin, was used to confirm the species identity. Furthermore, the isolate was confirmed as sequence type 9. The partial scm gene sequence harbored by the isolate was closely related to those of other two Sc strains. While this isolate did not possess the erm(A), erm(B), or mef(A), macrolide/lincosamide resistance genes, it was not susceptible to azithromycin: its susceptibility was intermediate. Even though human Sc bacteremia is rare, clinicians should be aware of this microorganism, as well as Pasteurella sp., Prevotella sp., and Capnocytophaga sp., when examining and treating patients with fever who maintain close contact with companion animals.

Impact of Sterilization Method on Protein Aggregation and Particle Formation in Polymer-Based Syringes.

The effects of sterilization methods on the storage stability of erythropoietin (EPO) in polymer-based syringes were assessed by quantifying protein oxidation, aggregation, and particle formation. Micro-particle counting and size exclusion chromatography coupled with a multi-angle light scattering detector demonstrated much lower levels of protein particles and aggregates for EPO stored for 12 weeks in steam-sterilized than in radiation (Rad)-sterilized syringes. Intermediate levels of damage were observed for EPO stored in ethylene oxide-sterilized syringes. HPLC analysis documented that the Rad-sterilized syringes caused increased oxidation of the protein during storage. In contrast, in the steam- and ethylene oxide-sterilized syringes EPO oxidation did not change. Analysis with electron spin resonance revealed that only Rad-sterilized syringes formed radicals in the syringe body, which persisted over the 12-week storage period. These results demonstrated that Rad-sterilization generated radicals in the syringes which in turn caused increased EPO oxidation, particle formation, and protein aggregation. Therefore, steam sterilization was shown to be a preferable sterilization method for the polymer-based syringe system when using biopharmaceutical drugs highly sensitive to oxidation, and particle formation and aggregation.

Low Leachable Container System Consisting of a Polymer-Based Syringe with Chlorinated Isoprene Isobutene Rubber Plunger Stopper.

UNLABELLED: A 36 month leachable study on water for injection in direct contact within a polymer-based prefillable syringe consisting of a cyclo olefin polymer barrel, a chlorinated isoprene isobutene rubber plunger stopper, a polymer label attached on the barrel, and a secondary packaging was conducted at 25 ± 2 °C and 60 ± 5% relative humidity. Through the various comparison studies, no difference in the leachable amounts was observed between this polymer-based prefilled syringe and a glass bottle as a blank sample reference by 36 months. No influence on the leachables study outcome was noted from the printed label and/or label adhesive or from the secondary packaging. In an additional study, no acrylic acid used as the label adhesive leachable was detected by an extended storage for 45 months at 25 ± 2 °C and 60 ± 5% relative humidity as a worst case. To obtain more details, a comparison extractable study was conducted between a cyclo olefin polymer barrel and a glass barrel. In addition, chlorinated isoprene isobutene rubber and bromo isoprene isobutene rubber were compared. As a result, no remarkable difference was found in the organic extractables for syringe barrels. On the other hand, in the case of element extractable analysis, the values for the cyclo olefin polymer barrel were lower than that for the glass barrel. For the plunger stoppers, the chlorinated isoprene isobutene rubber applied in this study was showing a lower extractable profile as compared to the bromo isoprene isobutene rubber, both for organic and element extractables. In conclusion, the proposed polymer-based prefillable syringe system has great potential and represents a novel alternative that can achieve very low level extractable profiles and can bring additional value to the highly sensitive biotech drug market. LAY ABSTRACT: A 36 month leachable study on water for injection in direct contact within a cyclo olefin polymer barrel and chlorinated isoprene isobutene rubber plunger stopper that has a polymer label attached to the barrel and is wrapped into a secondary packaging was conducted at 25 °C and 60% relative humidity. Through the various comparison studies, no difference in the leachable amounts was observed between polymer-based prefilled syringes and a glass bottle as a blank sample reference by 36 months. No influences on the leachables study outcome were noted from the secondary packaging. To obtain more details, a comparison extractable study was conducted between the cyclo olefin polymer and the glass barrel. In addition, chlorinated isoprene isobutene rubber and bromo isoprene isobutene rubber plunger stoppers were compared as well. As a result, no remarkable difference was found in the organic extractables for barrels. As for element extractable analysis, the values for the cyclo olefin polymer barrel were lower than that for the glass barrel. For the plunger stoppers, the chlorinated isoprene isobutene rubber applied in this study was showing a lower extractable profile as compared to the bromo isoprene isobutene rubber, both for organic and element extractables. In conclusion, the proposed polymer-based prefillable syringe system has great potential and represents a novel alternative that can achieve very low level extractable profiles and can bring additional value to the highly sensitive biotech drug market.

A strategy for the prevention of protein oxidation by drug product in polymer-based syringes.

UNLABELLED: 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.

Assessment of the effects of sterilization methods on protein drug stability by elucidating decomposition mechanism and material analysis.

The effects of different sterilization methods on the stability of highly sensitive protein drugs were assessed by elucidating mechanism involved in the process of protein decomposition. Results demonstrated that the steam sterilized syringes produced less protein oxidation compared with sterilization by the electron beam method. Electron spin resonance analysis showed that while considerably high levels of radicals were observed in the electron beam-sterilized syringes, no radicals were detected with steam sterilization. To identify the factor involved in protein oxidation, stability of the chemical composition of the syringe material was investigated using various analytical methods. Results showed that the syringe material itself was oxidized and two forms of oxidation products were identified with electron beam sterilization. Protein oxidation was shown to increase over time, and this was thought to be as a result of persistent exposure to the oxidized syringe barrel surface, which induced further protein oxidation. These results suggest that compared to electron beam sterilization, steam sterilization is a preferable method for the plastic prefilled syringe system, particularly for biopharmaceutical drug products that are highly sensitive to oxidization.

Functional evaluation and characterization of a newly developed silicone oil-free prefillable syringe system.

The functionality of a newly developed silicone oil-free (SOF) syringe system, of which the plunger stopper is coated by a novel coating technology (i-coating™), was assessed. By scanning electron microscopy observations and other analysis, it was confirmed that the plunger stopper surface was uniformly covered with the designed chemical composition. A microflow imaging analysis showed that the SOF system drastically reduced both silicone oil (SO) doplets and oil-induced aggregations in a model protein formulation, whereas a large number of subvisible particles and protein aggregations were formed when a SO system was used. Satisfactory container closure integrity (CCI) was confirmed by means of dye and microorganism penetration studies. Furthermore, no significant difference between the break loose and gliding forces was observed in the former, and stability studies revealed that the SOF system could perfectly show the aging independence in break loose force observed in the SO system. The results suggest that the introduced novel SOF system has a great potential and represents an alternative that can achieve very low subvisible particles, secure CCI, and the absence of a break loose force. In particular, no risk of SO-induced aggregation can bring additional value in the highly sensitive biotech drug market.