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Access to multiple analytical techniques is essential for fully characterizing complex protein formulations.
The purity (aggregates, host-cell proteins, host-cell DNA, etc.) and structure (sequence of amino acids, post-translational modifications, folding, etc.) of proteins directly impact the safety and efficacy of any formulated drug product based on them. Because proteins are generally produced as a mixture of similar but different biomolecules using living cells and subjected to extensive purification steps, variations in their structure and purity can occur from batch to batch.
The International Council for Harmonization (ICH) guidance Q6B, Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products (1) established guidelines for setting specifications for biologic drug substances, including determination of their composition, physical properties, and primary and higher-order structures, using appropriate methodologies. Comprehensive characterization of proteins is often challenging, however, given their complexity. In many cases, singular analytical methods cannot provide the complete picture for specific attributes. Orthogonal methods-different methods intended to measure similar attributes-are often necessary to provide independent confirmation of protein properties. Protein particle characterization is no exception.
The key when selecting orthogonal analytical methods is to choose technologies that overlap in complementary ways to provide a complete characterization of the protein in question, according to Kent Peterson, president and CEO of Fluid Imaging Technologies. The first step is to identify the information that is required for protein characterization and which techniques can provide that information-and the information that each cannot provide. “It is important to keep in mind that different techniques may provide the same information, but produce different results due to their varying technologies,” Peterson says.
Proteins, as well as all types of monoclonal antibodies and gene and cellular therapeutics, differ in terms of their structures, biological activities, and purity profiles. Consequently, while different sets of orthogonal technique strategies may be used for different biologics, true “toolbox” approaches or platforms may not be completely successful, according to Mike Merges, director of strategic growth for Catalent Biologics.
As a result, Catalent, when developing strategies for assay or method development and optimization, considers a combination of one factor at a time (OFAT) to define individual factors, at least initially, and design of experiment (DoE) to look at multiple and interacting factors. “The use of fractional factorial DoE as soon as is practical allows for a more rapid and robust development of a set of orthogonal methods,” Merges adds.
When it comes to particle analysis, Peterson notes that different techniques rely on different detection principles ranging from fairly basic (visual/microscopic inspection) to high-tech methods (resonant mass measurement [RMM], analytical ultracentrifugation [AUC]) and cover a wide range of particle sizes. “No single technique provides all of the answers. Two different techniques provide more data than just one,” he observes.
Effective sets of orthogonal methods “follow-the-molecule,” according to Merges. Timeliness, cost-effectiveness, and accuracy are other important factors, according to Peterson.
In addition, Merges notes that the ability to validate the techniques and transfer them from the development phase/lab to the quality control phase/lab or even to an outside contract research organization are essential.
“Validation is accomplished in a phase-appropriate manner,” Merges continues. The overall guidance for all phase-appropriate levels is ICH Q2 (R1) (2),
although different technical platforms (e.g., enzyme-linked immunosorbent assay [ELISA] or bioassay potency tests) may have specific levels of adherence to the ICH guidelines and/or other pieces of guidance, such as United States Pharmacopeia (USP) <1033> (3) and <1034> (4).
“The establishment of appropriate validation acceptance criteria should be based upon data-driven decisions. Those data are best generated via a pre-validation exercise conducted prior to the drafting of each phase-appropriate validation protocol,” he explains.
When it comes to particle characterization, Petersen points out that collection of imaging data is one of the accepted techniques for acquiring size, shape, and other morphological information used to differentiate protein aggregates from other particles in the formulation. “The nature of the particles in protein formulations and the small sample volumes pose a challenge in that throughput, accuracy, and specificity are met by very few technologies, however,” he comments.
Different techniques have different strengths and weaknesses for addressing these challenges. “For example,” Petersen observes, “particle count can be obtained by various particle analyzers, including single-function particle counters, light obscuration (LO) instruments, and flow imaging microscopes (FIMs). However, only some FIMs offer light and dark pixel thresholding that enables the detection of translucent proteins, while these proteins go undetected by other technologies.”
More specifically, LO provides particle count and concentration for large sample volumes. It provides the ability to analyze large volumes relatively quickly, has been around for a long time, and is seen as the first step in particle analysis, according to Peterson.
It does not capture morphological information about the particles contained in the sample, however, nor does it accurately detect or record translucent to semi-translucent particles, according to Peterson. “In LO, the signal from each particle is converted to a sphere of correlative size, and what is actually measured is the equivalent spherical diameter (ESD) of the generated sphere. We know, however, that not all protein particles are spherical in shape, so this method does not produce the most accurate size distribution,” he explains. LO is an inexpensive and quick method that has been in use for decades despite these limitations due mostly to the fact that it remains a USP compendial method.
FIM, on the other hand, enables the detection of particle sizes ranging from 300 nm-5 mm and reveals morphological characteristics of the particles imaged. In addition, FIM can detect translucent, semi-translucent, and opaque particles and allows for the differentiation of protein aggregates and non-proteinaceous particles.
Currently, light obscuration and membrane microscopy are required by USP regulations, and flow imaging microscopy is being rapidly embraced globally, according to Peterson. The combination of technological advancement and the industry’s acceptance of the need for a better method to fill the gap where methods like LO left a lot of unanswered questions-specifically for smaller micro- and nanoparticle analysis-have paved the way for newer methods to become widely accepted, he explains. “Better data from imaging flow microscopy provides morphological characteristics of the particles analyzed and answers these questions while providing a more detailed analysis,” he says.
Nanoparticle analysis lacks many orthogonal components. Historically, imaging in flow could not address this size range, according to Peterson, so information on size and shape and transparency through the imaging of nanoparticles had been missing. Newer technologies such as nano-flow imaging from his company are addressing this need. Automated membrane microscopy from HALO Labs and hyperspectral imaging from Spheryx are also offering promising solutions. “Until recently, there has been no imaging capability for flow imaging microscopy of particles in the nanometer size range. The FlowCam Nano imaging instrument from Fluid Imaging Technologies is an oil immersion flow microscopy technique that provides optical resolution down to the hundreds of nanometers,” Peterson says.
The breakthrough technologies described herein are examples of solutions being developed by instrument manufacturers in response to the needs of formulation development scientists for orthogonal analytical techniques for protein characterization. Bringing them to the market is increasingly difficult, however, according to Peterson. “There seems to be a growing lag time between commercial implementation and regulatory acceptance and requirements. These delays may be jeopardizing the timely, widespread use of technologies that can significantly contribute to the safely and efficacy of biotherapeutic drugs,” he asserts.
1. ICH, Q6B Test Procedures and Acceptance Criteria for Biotechnological/Biological Products, Step 4 version (ICH, 1999).
2. ICH, Q2(R1) Validation of Analytical Procedures: Text and Methodology, Step 4 version (ICH, 1994) (Complementary Guideline on Methodology dated 6 November 1996 incorporated in November 2005).
3. USP General Chapter <1033>, “Biological Assay Validation” (US Pharmacopeial Convention, Rockville, MD, 2010).
4. USP General Chapter <1034>, “Analysis of Biological Assays” (US Pharmacopeial Convention, Rockville, MD, 2012).
Vol. 42, No. 4
When referring to this article, please cite it as C. Challener, "Multiple Views Deliver Complete Characterization of Protein Particles" Pharmaceutical Technology 42 (4) 2018.