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Sep 15, 2009 (Vol. 29, No. 16)

Strategies for Successful Formulation

Tips on Streamlining Preformulation Projects, Particle Characterization, and Protein Aggregation

  • Characterizing Particles

    Click Image To Enlarge +
    Brightwell Technologies’ Micro-Flow Imaging system operates by capturing images from the sample as it passes through the flow cell’s sensing zone.

    One of those vendors is Brightwell Technologies, which will also be present at the IBC meeting. Deepak Sharma, Ph.D., senior research scientist, will host a workshop that describes how Micro-Flow Imaging™ (MFI) applies to biopharmaceutical formulations.

    Particle characterization is a standard method for measuring the stability of biotech products, particularly for protein aggregates and other species indicative of degradation, oxidation, or other undesirable chemical events.

    Conventional particle-analyzing instrumentation works best with opaque, spherical particles, but protein-based particles are highly irregular in shape, transparent to light, and usually unstable. For example, manual light microscopy and light obscuration, the default techniques for analyzing particulates in parenteral drug formulations, both have drawbacks that are becoming obvious as more biotech products hit the market.

    Manual methods are tedious, error-prone, and slow. Moreover, they consistently undercount protein-based particles that are almost always transparent and gel-like. Developed in the 1970s, light obscuration (LO) was designed to detect extrinsic particulates (e.g., glass, fibers, metal) larger than about 10 microns, in drug formulations. “Since LO was not designed to study intrinsic particles originating in bioformulations, its performance for these particle types is limited,” explains Dr. Sharma. Scattering and obscuration do poorly in sizing nonspherical or optically transparent species. When particles are detected, the information gleaned from them is limited.

    MFI, which was developed by Brightwell, and is currently under investigation at Wyeth, analyzes images of particles captured in succession as a sample stream passes through a flow cell. Size, shape, and intensity of each individual particle are measured, as well as the count and concentration of the entire particle population. MFI can also operate in a time-resolved mode to monitor dynamic processes.

    Maintaining a correlation between each particle’s morphological parameters and its image, aids in the classification of unique particle species, which may be isolated and characterized through software. MFI’s strength is monitoring protein aggregation or precipitation during formulation development, stability testing, production processes, and final-lot validation. It also detects and characterizes extrinsic particles such as air bubbles, silicone oil droplets, and foreign particles like glass, rubber, or metal.

    “The technology is already widely employed as an orthogonal technique in the formulation development phase of drug discovery by big pharmaceutical companies,” notes Dr. Sharma. “FDA has also started showing interest in MFI as an orthogonal technique.”

    Protein aggregation is a fundamental problem with implications in diseases, as well as biopharmaceutical formulations. One hallmark of Alzheimer’s disease, for example, is aggregation of amyloid plaques. Jennifer Laurence, Ph.D., professor of pharmaceutical chemistry at the University of Kansas, is planning to discuss her group’s investigations into factors and conditions that affect stability and aggregation, including mechanisms implicit in the formation of amorphous protein aggregates. “It’s an area where not a lot is known at the molecular level,” Dr. Laurence says.

  • The Disease-Aggregate Connection

    Dr. Laurence combines higher-resolution analytic techniques like solution NMR (nuclear magnetic resonance), combined with molecular engineering of proteins, to assess which regions of the protein are responsible for amorphous aggregation, and under what conditions. Low-resolution methods such as circular dichroism, Fourier-transfer infrared spectroscopy, and light scattering provide information on the system as a whole rather than specific regions.

    Solution NMR detects changes in chemical environment for resonant nuclei (typically uniformly labeled 13C- and 15N-enriched proteins) in response to changes such as heat or a rise in pH. NMR allows investigators to perturb the protein, observe which regions are most affected, and then do the same with a protein in which amino acids have been swapped out through protein engineering. The changes are then correlated with aggregation behavior.

    Parallel experimentation has become a popular method for numerous process- and development-related activities. Byeong Chang, Ph.D., CSO at Symyx Technologies, will talk about microscale, parallel techniques for formulation optimization. The Symyx core formulation module tests hundreds of liquid formulations automatically, including active ingredients, buffers, and excipients. The module has components for excipient compatibility, forced degradation, stability, and solid and liquid formulations.

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