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Sep 15, 2010 (Vol. 30, No. 16)

Protein Therapeutic Formulation Issues

Lack of Standards across Most Aspects of Process Impedes Efforts to Improve Quality

  • Stabilization of mAbs

    Surface tension and conformational stability are closely related. The later refers to maintaining a protein in its native structure while in solution. “Something that is conformationaly unstable will tend to lose its native structure more easily and that will lead to problems—that’s true of antibodies and proteins of any type,” explained Tom Leach, Ph.D., scientist II, formulations, MedImmune. 

    Native structure is required for structure-specific function of proteins. For example, enzymes have an active site that requires certain native structure to catalyze a reaction. Antibodies must bind to an antigen—this is structure specific as well. Adding excipients and determining which ones are best for stability is a science that has been developed over the past few decades. There’s a limited number of excipients that are recognized as safe for pharmaceutical use.

    Protein unfolding has been described as a struggle. On one hand, the protein wants to unfold and unravel in solution because it has thermal energy and wants to expand. On the other hand, the solution is exerting a surface tension that is trying to collapse the protein.

    A protein trying to unfold is expanding against its own interface or a protein-water interface. “The interaction parameters are measuring the interaction of the excipients with the protein at this protein-water interface,” said Dr. Leach. Preferential exclusion indicates the excipient prefers to be excluded from that interface—an absence of interaction. Excipients that are preferentially excluded are usually the best stabilizers.

    Some of the best-studied excipients are sugars, which tend to result in a more native protein structure. “If you add a little sugar to most proteins, you achieve some improvement of stability.”

    One important measurement is the unfolding temperature—the temperature at which a protein in solution unfolds when under temperature stress. The addition of excipient adds protein stability over increased temperatures. “This can be somewhat dramatic. To improve stability over 10 degrees, one can substantially increase the shelf life for biopharmaceuticals,” Dr. Leach stated.

  • Aggregates and Subvisible Particles

    Interest in protein aggregates was intensified when patients treated with therapeutic proteins became nonresponders. “Various researchers began to realize that maybe protein aggregates were the problem,” explained John Carpenter, Ph.D., professor of pharmaceutical biotechnology at the University of Colorado School of Pharmacy. “We realized the sweet spot for aggregates was in the subvisible range—probably around 10 microns.”

    This is an important number, because all parenteral products have to be assayed for particles greater than 10 microns.

    There is also interest in particles less than 10 microns as they may provide interesting quality product attributes that are not being analyzed nor reported in FDA filings. This has given rise to several issues: what is the smallest particle to worry about? is it a parameter that can be controlled? and are these particles an important issue for patient safety?

    “The answer is, we’re not sure. If nothing else, it’s a product-quality issue and should be addressed.”

    Although there is a lot of analytical work being done on protein particles, there is no definitive answer as to the best method. While particle counting is not difficult, it does have technical challenges that depend on factors like sample handling, and user experience.

    Microflow imaging is becoming a popular method to study protein particles. In this technique, a microscope takes digital pictures of a field as fluid flows through it. It counts and averages the number of particles, which provides a distribution of the number of particles versus diameter.

    A recent innovation by Graham Milne of Amgen uses video analysis on spun vials or syringes. The video of the particle behavior (sinking or floating), with their size and shape, enables noninvasive counting. This is helpful since all products require 100% visual inspection upon manufacture.

    “This method may allow people to have quantitative data across the visible and subvisible, and may eliminate the arbitrary absolute that if you can see it, you can’t use it. By looking at the subvisible particles, companies gain valuable data that helps them to understand visible particles in the context of everything,” Dr. Carpenter noted.

    Over the next year, there will be several in vitro and animal-based assays to analyze various aggregates and degraded proteins, he reported.

    “In therapeutic proteins, we always worry about chemical modifications and particles are part of that.” However, he says, “it will be awhile before we really understand how to measure and control particles in products as a quality attribute. I don’t see anyone setting hard specifications for particles smaller than 10 microns for a while.”


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