There is a significant challenge posed by protein aggregation and the presence of extrinsic particles in biopharmaceutical products. Developers are discovering that drug impurities and protein aggregation arise in numerous phases of formulation production, development, and quality control with technological developments in particle analysis.
The aim is to make sure that pure and safe biologics are produced, which can be supplied and shipped to patients while protecting their health and safety.
Numerous particle analysis technologies which are frequently utilized by biopharmaceutical developers can identify proteins and different particles at the sub-micron level but do not always identify bigger particles which may be found in a sample, for instance silicone particles, fibers, glass, or protein aggregates.
A flow imaging microscope, the FlowCam 8100, images, analyzes and counts particles in the range of 2 µm to 1 mm. It can ensure that a wider range of particles in a sample are analyzed when it is utilized through several stages of drug development as an orthogonal detection method. This enables drug developers to see the complete picture of what is contained in their product.
In order to analyze and count PSD standard samples at a range of sizes, William Bernt of Particle Characterization Laboratories, Inc (Novato, CA) employed three instruments in his study. He released a poster in 2017 entitled ‘Screening Biopharmaceuticals with Flow Imaging; Finding the Missing Fraction’ which contains the following excerpt.
Sub-visible proteinaceous aggregates are often a degradation product that can be a major factor in limiting the shelf life and efficacy of biopharmaceuticals. These aggregates may affect product manufacturability, bioactivity, and absorption rate. More importantly aggregates may bring about immunogenicity in the patient resulting in a loss of drug efficacy, patient discomfort or even death.
William Bernt, Particle Characterization Laboratories, Inc.
Bernt continued, "Establishing the presence of such particles is therefore of paramount importance when developing products for infusion or subcutaneous injection. We show how two commonly used particle sizing methods, dynamic light scattering (DLS) and nano-particle tracking (NTA), can easily fail to detect sub-visible micron-sized aggregates even at concentrations ten times greater than those specified by USP 788/787. Furthermore, we demonstrate how Flow Imaging Microscopy (FIM) plays a critical role in detecting, characterizing, and enumerating these sub-visible particles. Data is also presented on a commercially available 50 nm liposomal product.”
Figure 1. Instruments used in Bernt study. Yokogawa Fluid Imaging Technologies replaced the FlowCAM VS-1 with newer model FlowCam 8100 in 2016. Image Credit: Yokogawa Fluid Imaging Technologies, Inc.
The following method was employed by Bernt for sample preparation:
- Make up a 10 µm PSD standard (Thermo Scientific Corporation) suspension at 120,000 particles per ml (20X UPS-788 limit).
- Make up a 25 µm PSD standard (Thermo Scientific Corporation) suspension at 12,000 particles per ml (20X USP-788 limit).
- Add 4.5 ml of each suspension to a new Falcon tube to dilute the concentration of each standard by half (10X the USP-788 limit).
- Add 1 ml of a 10 wt% 186 nm NIST traceable PSD standard (final concentration 1wt % 186 nm PSD).
- Use the FlowCam VS-1 10X FC100 to analyze the sample neat.
- Dilute the sample 1:5,000 for the DLS analysis.
- Dilute the sample 1:50,000 for the NanoSight analysis.
Three different sizes of particle size distribution standards (186 nm, 10 µm, and 25 µm) were then analyzed by each instrument after being incorporated into one sample.
The DLS instrument detected an average particle diameter of 186 nm. This demonstrated that the instrument could not identify the 10 µm or 25 µm particles and only the 186 nm NIST traceable PSD standard particles were detected, as seen in Figure 2.
Figure 2. Dynamic Light Scattering Data. Image Credit: Yokogawa Fluid Imaging Technologies, Inc.
The NTA instrument detected an equivalent average particle diameter of 185.4 nm which showed that the NanoSight could also not detect the 10 µm and 25 µm PSD standard particles, as shown in Figure 3.
Figure 3. Nano-Particle Tracking Analysis Data. Image Credit: Yokogawa Fluid Imaging Technologies, Inc.
FlowCam Sees Larger Particles
The data exhibits peaks at 10 µm and 25 µm in the sample analysis which was carried out on the FlowCam, as shown in Figure 4. This indicates that although this FlowCam model does not identify particles at the nanometer level, it effectively images, detects and counts particles which are 2 µm and above.
The data also shows outliers that could signify extrinsic particles found in the sample. These individual particles can be identified thanks to the FlowCam’s imaging capabilities.
Figure 4. Flow Imaging Data. Image Credit: Yokogawa Fluid Imaging Technologies, Inc.
Figure 5. Intrinsic and extrinsic particles imaged by the FlowCam: protein aggregates, glass shards, silicone oil droplets, and other particulate. Image Credit: Yokogawa Fluid Imaging Technologies, Inc.
- Just because DLS and NTA don’t report micron-sized particles does not mean that they are not there.
- Flow Imaging Microscopy is crucial for evaluating biopharmaceutical formulations.
- Flow Imaging Microscopy augments other instruments which are designed to specifically characterize the sub-micron size component.
Produced from materials originally authored by Yokogawa Fluid Imaging Technologies, Inc.
This information has been sourced, reviewed and adapted from materials provided by Yokogawa Fluid Imaging Technologies, Inc.
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