From the Editor: Aggregate Knowledge

At the American Association of Pharmaceutical Scientists (AAPS) Annual Meeting in San Antonio last fall, several speakers shed light on one of the challenges of formulating injectable proteins: adding preservatives.

Laura Bush

Michael Akers of Baxter explained that no single antimicrobial preservative agent is ideal. Propylparaben, for example, is the only agent that is consistently effective against gram-positive and -negative bacteria, yeast, and molds, but it is not very soluble, and European regulators prefer to avoid it because of the risk of anaphylactic shock. The most common antimicrobial preservatives, benzyl alcohol, phenol, and M-cresol, all generally work, but they come with complications.

No single preservative agent is ideal from a physico-chemical or safety standpoint, and requirements to pass preservative effectiveness tests (PETs) vary. The United States Pharmacopeia requires that preservatives have a bacteriostatic effect, whereas European Pharmacopeia (EP) standards demand bacteriocidal formulations. Benzyl alcohol, at the 0.9% concentration levels that formulators tend to prefer, often does not meet EP criteria for log reduction values unless the formulation has other properties (e.g., low pH, or other antimicrobial components) that synergistically contribute to meeting these criteria.

Preservatives pose other complications: often they are incompatible with process materials such as tubing and filters, and they tend to bind with other formulation ingredients such as polysorbate 80 and cyclodextrins. The biggest problem, however, is that preservatives are often incompatible with the proteins themselves, leading to protein aggregation, which can cause immunogenic responses. Ironically, the effort to protect patients could actually harm them.

Determining the levels and types of aggregation is not a simple matter. Mary Cromwell of Genentech explained the strengths and weaknesses of various analytical methods for detecting aggregation. Because no single method is sufficient, the regulatory authorities generally expect to see several orthogonal methods.

The mechanisms and kinetics of preservative–protein interactions are not fully understood either. At the AAPS session, Eva Chi of the University of Chicago presented results of two studies done at the University of Colorado, on the benzyl alcohol–induced aggregation of a recombinant human interleukin-1 receptor antagonist (rhIL1-ra).

The team used a slew of analytical methods to examine the structure, conformational dynamics, and stability of the aggregates, as well as the benzyl alcohol–protein interactions. They found that the benzyl alcohol bound weakly to hydrophobic sites on the protein, increasing conformational mobility; as the numbers of partially unfolded species increased, so did aggregation. Adding sucrose decreased aggregation, apparently by helping maintain the native protein conformation.

The complexity of these issues makes it clear why protein aggregation has been receiving more attention recently. AAPS held open fora on this topic at the 2005 and 2006 National Biotechnology Conferences, and the University of Colorado hosted a protein aggregation workshop last September. Such meetings are important. A lot is known about protein aggregation, but there is much more to learn, and we need to ensure that new and experienced formulators alike are aware of the challenges and know the best ways to handle them.

Laura Bush is the editor in Chief of BioPharm International, lbush@advanstar.com