AAPS, San Antonio, TX (Oct. 31)—As biologic-based drugs become an increasingly larger part of pharmaceutical product portfolios, strategies for optimizing their formulation become increasingly important. A session at this year's annual meeting of the American Association of Pharmaceutical Scientists (AAPS, Arlington, VA, www.aapspharmaceutica.com) addressed a key step in this process: selecting an antimicrobial preservative.

Approximately 25% of protein products currently on the market contain antimicrobial agents, said Michael Akers, senior director, pharmaceutical research and development at Baxter Pharmaceutical Solutions LLC (Bloomington, IN, www.baxter.com) and editorial advisory board member of Pharmaceutical Technology. Akers spoke at the AAPS session, “The Impact of Preservatives on Physicochemical Properties of Therapeutic Proteins.”

M-cresol, phenol, and benzyl alcohol are the most widely used antimicrobial agents used in protein multi-dose products. They are generally used as single agents, although insulin-protamine suspension products, for example, contain both m-cresol and phenol.

“There are several issues to take into consideration when selecting an antimicrobial agent,” explained Akers. “First is understanding that there are potential incompatibilities with the protein itself, as well as potential incompatibilities with processing materials (tubing and filters), other formulation ingredients (polysorbate 80 and other polymers), and packaging materials (rubber).”

Another key challenge, explained Akers, is to find a globally acceptable agent, particularly given the differences in acceptance-test criteria in preservative-efficacy testing (PET) in the United States Pharmacopeia(USP) and the European Pharmacopoeia (EP). PET evaluates the effectiveness of preservative systems in the final product (formulation and package) when microbial agents are inoculated in the product. It is a method of demonstrating the effectiveness of the preservative throughout the shelf life and use of the product.

“EP test acceptance criteria are much more stringent than USP criteria,” explained Akers, with the EP requiring bacteriocidal formulations while the USP basically requires bacteriostatic formulations. He also offered some observations on the choice of antimicrobial preservatives that are used in formulations in Europe. While benzyl alcohol is the preferred antimicrobial preservative in Europe, concentrations of 0.9% often will not meet EP’s A criteria, but concentrations of 1.2–1.5% will. Phenol (0.5%), m-cresol (0.3%) or chlorocresol (0.25%) are acceptable, and the parabens are generally not preferred because of potential concerns for anaphylactic shock.

Methods of detection for protein aggregation

The choice of analytical method to detect protein aggregation is another key consideration. For protein therapeutics, the presence of aggregates is typically considered to be undesirable because of the concern that they may lead to an immunogenic reaction or may cause adverse events on administration of the drug product.

“Preservatives frequently cause aggregation in formulations, and several methods exist to detect aggregation, each offering advantages and disadvantages” said Mary Cromwell, scientist and senior group leader of Genentech, Inc. (South San Francisco, CA, www.gene.com) and committee chair of the AAPS Focus Group on Protein Aggregation and Immunogenicity.

Size-exclusion chromatography (SEC), a method in which particles are separated based on their hydrodynamic volume, is the industry workhorse for detecting aggregation, but dilution onto the column may cause dissociation, and large aggregates may not enter the matrix.

SDS (sodium dodecyl sulfate)-based techniques such as capillary electrophoresis (CE) or polyacrylamide gel electrophoresis (PAGE) may be used as well, and offer versatility since the method is not protein specific and is sensitive to trace quantities.

Analytical ultracentrifugation (AUC), fluid flow fractionation (FFF), light-scattering techniques, light obscuration, and the Coulter counter are other methods that are good for particle detection, said Cromwell.

Evaluating benzyl alcohol–induced aggregation

Eva Y. Chi, postdoctoral research fellow at The University of Chicago (www.uchicago.edu), outlined research done while at the University of Colorado (www.colorado.edu) for evaluating the mechanism and kinetics of benzyl alcohol–induced aggregation of rhIL1-ra, a recombinant human interleukin-1 receptor antagonist.

Chi outlined that benzyl alcohol, a commonly used antimicrobial agent in therapeutic protein formulations, accelerates the non-native aggregation and precipitation of rhIL-1ra by increasing the population of aggregation-prone, prenucleating species, which eventually react to form insoluble precipitates. Kinetically, nucleation rates were found to increase by about four orders of magnitude in the presence of 0.9% benzyl alcohol. Once insoluble aggregate nuclei are formed, however, the rate at which nuclei grow into larger aggregates was not significantly affected by the presence of benzyl alcohol. Sucrose was found to block aggregation of rhIL1-ra by favoring compact native state structures that were not aggregation-prone. Addition of sucrose sharply decreased aggregate nucleation rates while having little effects on the rate of aggregate growth.