Another major theme at the meeting was protein aggregation. No product can be guaranteed 100% free of aggregates. Hanns-Christian Mahler, Ph.D., head formulation R&D biologics at F. Hoffman-La Roche(www.roche.com), reviewed the types and causes of aggregation, pointing out that issues as diverse as transportation (bumps on the road) and the use of piston pumps and certain types of packaging can trigger formation of aggregates.
Manufacturers have to be aware of aggregation because it may become a safety issue through affecting pharmacokinetics or causing immunogenicity. “There is a huge debate over the immunogenicity issue,” Dr. Mahler said. “There is a need for further solid research on this.” Certainly the regulatory authorities take an interest in aggregation, having requirements for clarity and limits upon even subvisible particles.
Dr. Mahler added that there are ways of controlling aggregation—for instance, shaking a solution is less likely to cause aggregation than stirring it. He believes that a battery of methods is needed to investigate the full range of aggregation, by size (from monomer to visible aggregate particle), with size exclusion chromatography being the workhorse, although there are also roles for dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and ultrasonic resonance.
Muppalla Sukumar, Ph.D., research advisor at Eli Lilly (www.lilly.com), noted that aggregation often goes undetected and the underlying mechanisms are complex. Measuring aggregation is challenging because of the range of sizes, and now regulatory authorities are expecting the application of orthogonal methods to tackle the problem.
“It is not just important to determine the amount of aggregation—its composition matters, too,” Dr. Sukumar said. He believes that DLS is good for detection of low levels of large aggregates but bad for resolution. AUC with sedimentation velocity, however, can give a chromatogram-like result that helps to resolve different sized aggregates. In summary, SEC has high precision but can lead to loss of aggregate because of dilution effects or interaction with matrix; AUC is accurate but has low precision and is not QC compliant; while DLS is highly sensitive for large aggregates.
“SEC will be the control method for some time to come. AUC and DLS are not robust or easy to validate for lot release. They are best used in drug development,” Dr. Sukumar concluded, citing a case study in which AUC had been used to trace the source of aggregation in a purified monoclonal antibody to a neutralization step. Significantly, the aggregate was detected by AUC and not SEC. Further, the aggregate did not turn up in the API, so it was not an intrinsic property of the molecule.
Dave Thomas, technical sales director at Brightwell Technologies (www.brightwelltech.com), described the options for assessing formulation stability and protein aggregation in terms of visible and subvisible particle formation. There is light extinction/obscuration that can be used to calculate the size of particles, but it is not suitable for translucent or heterogeneous particles. The sample may need to be diluted, which could induce aggregation or creation of air bubbles. Manual microscopy is another option but it is labor intensive. Brightwell’s MFI™ (micro flow imaging) Flow Microscopy system is a potential solution, according to Thomas.
“MFI combines the speed and convenience of particle counting with the visual insights and material insensitivity provided by manual microscopic analysis,” Thomas noted. With MFI, a volumetric section of fluid is investigated so that particles can be counted, sized, and imaged. It can also deal with small sample volumes and accommodate higher particle concentrations than light extinction. “MFI is best suited to micrometer-sized particles. It is applicable to contamination detection/classification and continuous monitoring of suspensions.
“Protein aggregates are challenging because they are highly transparent, fragile, and have an irregular shape and size,” said Thomas, who added that experiments at Brightwell showed that the MFI approach can deal with these issues. In one example, it compared MFI with obscuration to check the effectiveness of filtration with respect to particle removal and the formation of protein aggregates.
“MFI was one to two orders of magnitude more sensitive, showing particles that get through the filter, or reform after filtration,” Thomas explained.
Therapeutic proteins may have to be modified to achieve their full therapeutic potential. One new approach is the albufuse™ technology developed by Novozymes (www.biopharma.novozymes.com). “New therapeutic proteins can be unstable, immunogenic, or have a short half-life or low bioavailability” said Dave Mead, Ph.D., business development director, Novozymes Biopharma UK. “Our albufuse albumin fusion system fuses therapeutic protein with recombinant albumin at the molecular level endowing the protein of interest with the natural properties of the latter.”
Albumin is an abundant and benign carrier protein with a circulatory half life of 19 days and as such, it is a great vehicle for delivery of active-drug candidates, Dr. Mead noted. “Fusion to albumin does increase the circulation time and in vivo time and so endows a longer half life.”