The Truth Behind Protein Aggregation in Prefilled Syringes

Prefilled syringes are widely used as container closure systems for biologic drug products such as monoclonal antibodies, cytokines, and vaccines (1). The advantages of prefilled syringes compared with vials include the ease of use, which enables safe and convenient self-administration; reduction of medication errors; and product differentiation to name a few (2).

For protein therapeutics, however, the stability of the formulation in prefilled syringes can be negatively impacted by interactions between the drug molecules and components of the container-closure system. For example, protein may absorb to silicone oil, which is often used in prefilled syringes (3). It is, therefore, important for manufacturers to pay close attention to the material characteristics of the prefilled syringe because protein adsorption can lead to protein aggregation, which then presents risks of immunogenicity. Fran DeGrazio, vice-president of scientific affairs and technical services at West Pharmaceutical Services, spoke with BioPharm International about protein aggregation in prefilled syringes.

Causes of Protein Aggregation

BioPharm: What components/materials in prefilled syringes can cause protein aggregation?

DeGrazio (West): Protein aggregation can be caused by many different components/materials within a prefilled syringe that are inherent to the general manufacturing and the final drug product state such as: the siliconized glass barrel and subsequent formation of silicone oil droplets (4), residual tungsten deposits remaining within the conical end of the glass barrel after manufacturing (5), potential leachates from the elastomeric plunger and needle shield/tip cap (6), as well as acrylic acid leachates from adhesive that attaches the needle to the conical end of the glass barrel (7). These are the most significant causes relating directly to a typical glass prefilled syringe system.

BioPharm: Can you tell us more about the interactions between the drug product and the components/materials of the prefilled syringe and how they can affect the safety and efficacy of the protein formulation?

DeGrazio (West): Non-uniform silicone coating within a prefilled syringe system is a key source of protein-silicone oil interactions within a glass barrel (8). Silicone oil can be bound to the barrel or be found as free-floating silicone oil droplets. The rate of adsorption onto the silicone oil is dependent upon the amount of surface area with which the protein has to adsorb (5). The effects of silicone oil on protein adsorption tendency and its kinetics are a precursor to the development of protein aggregates (5).
Tungsten is introduced into the system during the formation of the needle hole at the cone of the barrel during manufacture. Residual tungsten oxide vapor deposits--tungsten trioxide (WO3)-can result from this process and interact with the drug product to create polyoxoanionic tungstate complexes that induce protein aggregation (6). The complexes that are formed are capable of physically adsorbing and adhering to the surface of proteins (7). This complexation with the surface of a protein can alter the energetics of the surface increasing the likelihood to aggregate (9). 

Rubber components are composed of many different constituents. Some of these components have the potential to leach directly into a drug product. As an example, some elastomer components may use peroxide to cure or assist in the crosslinking of the elastomer formulation. These peroxide-cured rubbers could leach breakdown products (i.e., volatile organics) of the cure system that have the potential to initiate oxidative damage to a biologic product.

The adhesive used to attach the needles of a prefilled syringe to a glass barrel has been shown to be a source of acrylic acid leachates. It has been demonstrated that these leachates can interact with therapeutic proteins. After being modified by acrylic acid, susceptible amino acids can form adducts that alter the conformation of a protein’s tertiary structure. The impact can affect the isoelectric point as well as the protein’s surface charge distribution leading to potential aggregation that can decrease the therapeutic efficacy of the drug product (10).

Particles due to glass delamination typically do not occur with prefilled syringe systems. Glass delamination is typically found with vial systems. The vial manufacturing process uses greater heat input thus leading the surface of the glass to become more reactive.

Each of the drug product-component interactions described have the potential to induce protein aggregation. Depending upon the level and severity of the protein aggregates formed, there is an inherent risk to patient safety and the quality of the drug product. Aggregation can suppress the efficacy of a drug product by altering the native structure of the active pharmaceutical ingredient. The tendency of the biologic to aggregate and the body’s response in eliciting antibodies towards those aggregates correlates with each other (10).

Studies have shown that aggregate complexes with molecular weights greater than 10 kDa are sufficient to induce the formation of antibodies (11). It is essential to minimize protein aggregation prior to patient administration to limit potential immunogenic responses that can limit the drug’s efficacy or even potentially harm the patient. This is where the primary package/delivery system itself and its interaction with the biologic may have a direct impact on patient safety.

Compatibility Assessment

BioPharm: How do you assess compatibility of a protein formulation with the prefilled syringe and its components/materials?

DeGrazio (West): From the perspective of a formulation scientist, forced degradation studies are typically performed to assess the compatibility of a drug product with any container closure system. Because it is known that tungsten, silicone oil, adhesive, and transition metals are the key elements within a prefilled syringe system that have the highest capacity to induce drug product instability, spiking studies with each item at varying levels are carried out over specific durations.

As prefilled syringes can be siliconized using either a spray-on or baked-on technique, the level of siliconization and the degree of homogeneity within each glass barrel is also assessed. The amount of siliconization that is acceptable can be correlated to the results obtained from spiking studies. Similarly, extractable and leachable studies of the prefilled syringe to be used for the drug product are executed to establish limits for the tested leachables.

The other aspect that is crucially important is functionality or performance of the syringe system. Syringeability for the application must be understood. Time and force are critical factors that bear a direct impact on the patient. The amount of silicone oil, the viscosity of the drug product, and the plunger design are all variables that can directly impact syringeability. An understanding of the final intended method of delivery, whether a manual injection, an autoinjector, or some other delivery device approach must be understood to best qualify the prefilled system.

The Parenteral Drug Association (PDA) released Technical Report No. 73, which elucidates the prefilled syringe user requirements for biotechnology applications (12). The considerations are broad and range from human factors to container closure integrity requirements.

Ensuring Protein Stability

BioPharm: How do you ensure protein stability throughout the shelf-life of a drug product, as well as during storage and transportation?

DeGrazio (West): Through a combination of biologic drug formulation, primary package, and delivery system considerations, one can minimize the potential for aggregation and improve drug product stability. A robust formulation is developed using high-throughput screening via a design of experiment approach. Freeze/thaw studies and agitation studies are performed, which can simulate the effects that can be seen in transportation.

With respect to the prefilled syringe as the primary package, an improved understanding of the materials and alternatives that are available can be one way that these challenges are mitigated. For instance, the biologic drug has direct contact with the elastomeric components within a prefilled system. This direct contact exaggerates the potential impact of the syringe materials of composition in comparison to a vial/stopper system. One way to mitigate extractable migration from the elastomeric components to the biologic drug product is through the use of components that use barrier films or coatings. Fluroelastomer film coatings are commonly used as a barrier between the drug and elastomer to minimize leaching potential. Of course, it is still very important to understand the characteristics of the materials to which the film or coating is applied, as one must understand what extractables could migrate over the shelf-life of the product, albeit in much lower quantities than may have occurred with an uncoated plunger. These films also have the added benefit of providing lubricity to the plunger, helping to minimize the use of loose silicone oil in the system.

In some cases, polymeric syringe systems produced from polycyclic olefins are coupled with fluroelastomer coated plungers. Some combinations, such as the Daikyo Crystal Zenith insert needle system, have the benefit of being free of silicone oil, adhesives, and tungsten because these materials are not part of the manufacturing process or of the final syringe system.

Every drug product formulation interacts with the final, primary packaging system differently. The appropriate container closure system has to be evaluated on a case by case basis. There is no ‘universal’ formulation or packaging system that is optimal for every biologic; therefore, stability data are the only true indicator to support a drug’s physiochemical quality throughout its shelf-life.

References

1. G. Walsh, Eur. J. Pharm. Biopharm. 55 (1) 3-10 (2003).
2. M. Adler, “Challenges in the Development of Pre-filled Syringes for Biologics from a Formulation Scientist’s Point of View,” Amercian Pharmaceutical Review, Feb. 1, 2012. 
3. A Siew, Pharm. Tech. 40 (4) 24-26 (2016). 
4. N. Dixit et al., Pharm. Sci. Tech. 13 (4) 1116-1119 (2012).
5. L. Liu et al., Invest. Ophthalmol. Vis. Sci. 52 (2) 1023-1034 (2011). 
6. A. Sedle et al., Pharm. Res. 29 (6) 1454-1467 (2012). 
7. E.Y. Chi et al., Protein Sci. 12 (5) 903-913 (2003). 
8. N. Dixit et al., Pharm. Sci. Tech. 13 (4) 1116-1119 (2012). 
9. J.S. Bee et al., J. Pharm. Sci. 98 (9) 3290-3301 (2009).
10. D. DeGrazio, PDA J. Pharm. Sci. Tech. 69 (2) 219-235 (2015).
11. A.S Roseberg, AAPS J. 8 (3) E501-E507 (2006).
12. PDA, Technical Report No. 73, Prefilled Syringe User Requirements for Biotechnology Applications (2015).

Article Details

BioPharm International
Vol. 29, No. 11
Page: 50-52

Citation

When referring to this article, please cite as A. Siew, "The Truth Behind Protein Aggregation in Prefilled Syringes," BioPharm International 29 (11) 2016.