Aggregation Analysis of Therapeutic Proteins: Quantitative and Characteristic Analysis of Sub Visible Particles (SVP)
In the aggregation analysis of therapeutic proteins, we introduce the reasons why quantitative and characteristic analyses of SVP (Sub Visible Particle) are necessary, the various analysis methods of SVP, analysis results by Malvern Panalytical, particle characteristic analyses, and improvement examples.
Table of Contents
- Reasons for the necessity of quantitative analysis of aggregation and particle characteristic analysis
- Methods of SVP analysis for aggregate quantitative analysis and analysis results by Malvern Panalytical
- SVP Analysis Method 1: DLS, LD for wide-range following
- SVP Analysis Method 2: SEC, AUC excellent for quantitative evaluation of monomers to oligomers
- SVP Analysis Method 3: NTA, RMM method for submicron range following
- Particle characteristic analysis for aggregate quantitative analysis and improvement examples
- Related analytical instruments by Malvern Panalytical
Reasons for the Necessity of Quantitative Analysis of Aggregates and Particle Characteristic Analysis
Aggregates included in therapeutic proteins such as antibody drugs are not visually detectable. Therefore, quantitative evaluation using some analytical instruments to determine how much aggregation is included is necessary.
Moreover, particle characteristic analysis (Particle Characterization) is required to distinguish whether the detected particles are protein-derived, additive-derived (such as sugar), or other debris.
These particles often exist between visible and completely invisible areas, referred to as Sub visible particles or abbreviated as SVP. Below we describe various analyses of SVP and analysis results by Malvern Panalytical.
SVP Analysis Methods for Quantitative Analysis of Aggregates and Analysis Results by Malvern Panalytical
Pharmacopoeias of various countries and regions, including the FDA, recommend SVP evaluation using multiple instruments. Unfortunately, each method has its pros and cons, and analyses with a single method entail risks of errors. Malvern Panalytical provides a lineup of multiple instruments in the field of SVP analysis to propose the optimal method according to the situation.
Additionally, the FDA issued guidelines in 2014 regarding the “evaluation of immunogenicity of biopharmaceuticals,” and one of the items mentioned is the measurement of protein aggregates.
SVP Analysis Method 1: DLS, LD for Wide-Range Following
DLS (Dynamic Light Scattering) and LD (Laser Diffraction/Scattering Method) are qualitative but suitable methods for indicating the overall distribution of proteins due to their wide dynamic range.
The following graph shows the particle size distribution of a marketed therapeutic antibody (red) and research IgG (green) by DLS. The therapeutic antibody shows a sharper distribution, suggesting that it has been finished to a size with consistent shape.
SVP Analysis Method 2: SEC, AUC Excellent for Quantitative Evaluation of Monomers to Oligomers
Currently, there is no principle that covers all ranges for quantitative evaluation.
For principles that enable quantitative evaluation from monomers (about 10 nm; in the case of full-length antibodies), SEC (Size Exclusion Chromatography) or AUC (Analytical Ultracentrifuge) are available.
Among them, SEC can easily calculate the monomer/oligomer ratio. Adding a light scattering detector can determine detailed molecular weight differences.
SVP Analysis Method 3: NTA, RMM Method for Submicron Range Following
The submicron range (0.1 – 1 μm), where SVPs are considered particularly abundant, has only recently become quantitatively detectable, with methods like NTA (Nanoparticle Tracking Analysis) and RMM (Resonant Mass Measurement) Method being applicable.
The submicron range is not only easy to evaluate quantitatively due to the abundance of presence but is also considered to be significantly related to immunogenicity in recent years, hence, the FDA strongly recommends its measurement.
The graph shows results measured by Archimedes (RMM Method), demonstrating a clear difference in SVP amount between stable and unstable formulations.
Particle Characteristic Analysis for Quantitative Analysis of Aggregates and Improvement Examples
The FDA also recommends analysis of particle characterization (Particle Characterization) and suggests “at least 2 orthogonal analytical techniques” for analysis.
The principle of Particle Characterization begins with “the particle existed”. In other words, the subsequent improvement method differs depending on what the detected particle is.
For example, if the particle is protein-derived, the protein or formulation should be reviewed, whereas if it is a fragment of cellulose, the filter or chromatographic column materials should be reconsidered. In this case, if image analysis and Raman imaging function are available, it is possible to perform particle characterization that is difficult to judge based on shape alone.
Case 1: Improved Manufacturing Process for Therapeutic Antibodies
Case 2: Protein Aggregates and Silicone Oil Droplets in Injectables
The most common case of materials other than protein being detected as particles is the prefilled syringe formulation (hereinafter referred to as prefilled), which has particularly increased in recent years.
Prefilled syringes are formulations sold with protein solution filled inside the syringe; however, the insides of the syringe are coated with a lot of oil (such as silicone oil) to facilitate smooth piston movement. While silicone oil itself is not problematic, recent reports indicate that this silicone oil promotes protein aggregation, highlighting the necessity of confirming the quantity of each.
Related Analytical Instruments by Malvern Panalytical
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Particle Size, Zeta Potential, Molecular Weight Measurement Instrument: Zetasizer Series Contributing to the research, development, and quality control of nanotechnology, the Zetasizer series can measure particle size, zeta potential, and absolute molecular weight, offering complex functions related to various particle measurements through simple operations. |
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Nano Tracking Particle Sizing Instrument: Nanosight Series By tracking the Brownian motion of particles and measuring each particle, it provides high-resolution particle size distribution and particle concentration. |
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High Sensitivity, High Precision, High Performance Multi-Detector GPC/SEC System: OmniSEC With a maximized sensitivity multi-detector GPC/SEC system, it measures even dilute or low-molecular-weight samples with high precision and reproducibility. |
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Particle Image Analysis and Raman Spectroscopy Instrument: Morphologi 4-ID Analytical instrument applicable in pharmaceuticals, ceramics, batteries, and chemicals. It measures particle size and shape automatically, enabling significant difference measurements. |
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