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By Satish K. Singh and Hanns-Christian Mahler

Singh-finalHanns-Christian MahlerNonionic surfactants are commonly used in formulations for biotherapeutics. Added in small amounts, they generally protect the protein from physical degradation by air-water, solid (ice, container)-water, or even oil-water interfaces. Both adsorptive loss of the protein and any interface-induced aggregation or precipitation are prevented or minimized by the surfactant. The protection provided by the surfactant arises from its ability to rapidly interact or coat these interfaces, thus competitively preventing proteins from adsorbing, unfolding, and subsequently aggregating there. Surfactants may also preferentially interact with the hydrophobic surfaces of proteins and thereby prevent both a close approach and association of these surfaces, preventing self-association and subsequent aggregation.

To determine the need for and the amount of surfactant required, formulators therefore primarily use agitation (i.e., generation of an air-water interface) as a stress-factor. Surfactant levels in commercial products, whether liquid or lyophilized, range in the low tenths of mg/mL. The most popular surfactants are the polysorbates (polyoxyethylene sorbitan fatty acid esters), with poloxamers (PEO-PPO-PEO triblock copolymers) coming in a distant second in use.

The commonly used polysorbates (PS20 and PS80) are based on a primary fatty acid (lauric acid C12:0, and oleic acid C18:1, respectively) but also contain a significant proportion of other fatty acids (usually as esters but sometimes as free fatty acids). The polysorbates, due to their structure, can undergo auto-oxidation, cleavage at the ethylene oxide subunits, and hydrolysis of the fatty acid ester bond. However, degradation of polysorbates can lead to the formation of subvisible and visible particles in the final drug product due to the insolubility of free fatty acids. Also, a novel enzyme-catalyzed hydrolytic degradation pathway has been proposed, possibly arising from a lipase present in host cell proteins.

The importance of this excipient, coupled with the heterogeneity of the raw material, the relatively low concentrations at which it is used, and the complexity of the degradation pathways and of the degradation products has led to a resurgence of interest in all questions related to the use of polysorbates in biotherapeutics.

How does one quantify the amount of polysorbate in a product? The lack of chromophores in polysorbates has resulted in the development of nontraditional detection methods. There is no widely recognized or universal detection method, and absolute quantification on the basis of a certain part of the molecule (e.g., fatty acid content) can give variable results.

Should all degradation products be included in the quantification? It is likely that some of the polysorbate degradation products are still functional as protectants, in which case they perhaps should be included in the quantification. Considering the wide use of polysorbates across the biopharmaceutical industry, there is interest in determining whether the cascade of polysorbate degradation impacts proteins negatively. Are particles stemming from polysorbate degradation a concern (PDF)? Is the impact different for PS20 (versus PS80) due to the lack of an unsaturated fatty acid?

Many of the analytical and protein impact aspects will be the topics of presentations at the symposium How Stable Is the Stabilizer? Polysorbate Degradation and Impact on Biopharmaceuticals, at the upcoming 2015 AAPS National Biotechnology Conference.

Satish K. Singh, Ph.D., is a research fellow at Pfizer in the Biotherapeutics Pharmaceutical Sciences group. He has expertise in formulation, product, and process development activities for biologics and therapeutic vaccines, and is involved with regulatory aspects of biologics development.
Hanns-Christian Mahler currently leads Pharmaceutical Development & Supplies, Biologics EU at F.Hoffmann-La Roche AG. He is interested in parenteral product development and manufacture. Hanns-Christian was elected an AAPS Fellow in 2013.