Protein Aggregates and Immunogenicity

I work as a microbiologist within the Quality Control unit for a small biopharmaceutical company that manufacturers several protein‐based parenteral products. One of the benefits of working for a small company is your role usually has a broader scope than someone in a larger organization. In addition to validating the standard microbiological release tests for in‐process microbial enumeration, endotoxin and sterility, I have been heavily involved in validating and performing the USP <788> subvisible particulate test by light obscuration for our parenteral products. In recent years, this chapter has come under much scrutiny in its applicability, or lack thereof, to protein‐based parenterals.

One of the drivers for this scrutiny is the risk of immunogenicity to the patient that can be brought on by protein aggregation. Proteins may aggregate for various reasons during manufacture, filling, packaging, and after release. Because of this risk, the FDA has asked many in industry to detect and quantify aggregated protein in their protein‐based drugs and determine the risk, if any, of immunogenicity to the patient. There has been much communication between industry, the FDA, and academia on this issue with no clear consensus on how it should be addressed.

One reason for this lack of consensus is no two protein drugs are alike. The protein in Drug A may aggregate and/or be immunogenic while the aggregated protein in Drug B may not. Currently, the FDA has asked many biologics manufacturers to test for subvisible particles by light obscuration down to the ≥2 micron size (current USP sizes are ≥10 and ≥25 microns) since it is thought that aggregates can be detected in this 2‐10 micron range. It is known that using light obscuration to detect protein aggregation has many drawbacks. For one, protein aggregates are relatively translucent and will result in undercounting. Also, all particles and not just protein aggregates will be counted in this lower range. There is also a lack of an appropriate protein standard for instrument calibration and system suitability purposes. Using light obscuration also raises other questions. What is to be made of results caused by non‐protein particles? How much aggregation is acceptable? Should aggregation studies be part of clinical trial development? These questions and many others have added much difficulty to the validation and performance of the subvisible particulate test for protein‐based parenterals.

In the years since I first started validating subvisible particle counting by light obscuration for protein‐based products, I’ve realized that I’ve had to add validation steps to determine suitable degas and mixing methods for each protein product. And there may be more. Looking to the future there is much interest in micro‐flow imaging technology that is being used more and more by industry for characterization studies. This technology is rapidly becoming more sophisticated in providing an accurate assessment of protein aggregation and may one day become a part of USP<788>. Although the issue of protein aggregates and immunogenicity is challenging and there are often more questions than answers, it is exciting for me to be involved in the development of methods that provide further measures of the safety of the medicines being manufactured.


Karla Aberle M.S., RM (NRCM), QC Microbiology Analyst III, Sigma‐Tau Pharmasource, Inc., Indianapolis, IN. Ms. Aberle achieved her NRCM certification in 2008.


Copyright© National Registry of Certified Microbiologists. Reprinted from The Loop, 2010, Issue 2.

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