The challenge posed by protein aggregation and the presence of extrinsic particles in biopharmaceutical products is significant. With technological developments in particle analysis, developers are finding that protein aggregation and drug impurities arise in several phases of formulation production, development, and quality control.
The aim is to ensure that safe and pure biologics are created that can be delivered and shipped to patients while protecting their health and safety.
A number of particle analysis technologies often used by biopharmaceutical developers are able to identify proteins and different particles at the sub-micron level, but do not always identify larger particles that may be found in a sample, for example silicone particles, glass, fibers, or protein aggregates.
The 8000, a flow imaging microscope, images, analyzes, and counts particles in the range of 2 µm to 1 mm. When the instrument is used through several stages of drug development as an orthogonal detection technique, it can ensure that a greater range of particles in a sample are analyzed, which allows drug developers to observe the complete picture of what their product contains.
William Bernt of Particle Characterization Laboratories, Inc (Novato, CA) used three instruments in his study to analyze and count PSD standard samples at a range of sizes. Bernt 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.
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 50nm liposomal product.”
Figure 1. Instruments used in Bernt study. Yokogawa Fluid Imaging Technologies replaced the FlowCAM VS-1 with newer model FlowCam 8000 in 2016. Image Credit: Yokogawa Fluid Imaging Technologies, Inc.
The following technique was used by Bernt for sample preparation:
- “Make up a 10µm PSD standard (Thermo Scientific Corporation) suspension at 120,000 particles per ml (20X UPS-788 limit).
- Make up a 25µm PSD standard (Thermo Scientific Corporation) suspension at 12,000 particles per ml (20X USP-788 limit).
- Add 4.5ml of each suspension to a fresh Falcon tube. This diluted the concentration of each standard by half (10X the USP-788 limit).
- Add 1ml of a 10wt% 186nm NIST traceable PSD standard (final concentration 1wt% 186nm PSD).
- Analyze the sample neat through the FlowCam VS-1 10X FC100.
- 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 incorporated into one sample and were then analyzed by each instrument.
An average particle diameter of 186 nm was detected by the DLS instrument, showing that only the 186 nm NIST traceable PSD standard particles were detected and the instrument could not identify the 10 µm or 25 µm particles (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 demonstrating that the NanoSight equally could not detect the 10µm and 25µm PSD standard particles (Figure 3).
Figure 3. Nano-Particle Tracking Analysis Data. Image Credit: Yokogawa Fluid Imaging Technologies, Inc.
FlowCam Sees Larger Particles
In the sample analysis performed on the , the data demonstrates peaks at 10 µm and 25 µm (Figure 4). This suggests that although this FlowCam model does not identify particles in the nanometer level, it effectively images, detects, and counts particles that are 2 µm and larger.
The data additionally depicts outliers that could signify extrinsic particles found in the sample. The FlowCam’s imaging capabilities enable these individual particles to be identified.
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.
- “Flow Imaging Microscopy is a critical tool for assessing biopharmaceutical formulations.
- Flow Imaging Microscopy augments other instruments which are designed specifically to characterize the sub-micron size component.
- Just because DLS and NTA don’t report micron-sized particles does not mean that they are not there. Verify!”
This information has been sourced, reviewed and adapted from materials provided by Yokogawa Fluid Imaging Technologies, Inc.
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