Particulate matter found in injectable drugs is measured in the visible and sub-visible ranges. In the sub-visible range, measurements are generally made by liquid particle counter, or by microscopic analysis of the sample captured on a filter.
The filter test technique was introduced to eliminate the extra counts, due to the presence of silicone oil droplets introduced by stoppers, and barrels, and plungers of pre-filled syringes. Although the inner silicone oil droplets are likely to be less of a health hazard than external contaminants, such as metal shards or fibers, they will be detected by liquid particle counters. Pre-filled saline syringes using silicone oil on the barrel and/or inner barrel wall will produce a number of supplementary particle counts because of the silicone oil emulsion droplets.
The syringe used for this particular research was the BD PosiFlush Normal Saline Syringe, 10 mL, Ref no. 306546, (Figure 1). Predictable flush installations and smooth stopper actions are facilitated by applying medical-grade silicone oil on the inside wall of the syringe barrel and to the stopper.
Measurements were taken of only the saline present in the syringes, and the mixture of saline and protein. The protein used was Athens Research & Technology, Immunoglobin G, Human Plasma, Lot no. IG2014-02, Cat no. 16-16-090707, lyophilized from 22.4 mL 20 mM phosphate buffer, pH 7.4, w/150 mM NaCal and 0.05% sodiumazide. The protein was dispersed in filtered PBS; Phosphate buffered saline tablet, Sigma Aldrich P4417-50TAB.
To start, the particle counts from the syringes were taken directly from the syringe without activation; by pushing in a sample tube via the outlet for the AccuSizer, or by removing the barrel and pouring the sample into a clean beaker, or with activation; by pushing the plunger driving the sample into a clean beaker.
The IgG was prepared at 0.1 mg/mL in filtered PBS and tested. Next 0.5 ml IgG was pipetted into 10 mL of either filtered PBS or saline from a pre-filled syringe. Measurements were made using the AccuSizer without dilution using 1 mL of sample, measured three times to verify repeatability.
The Entegris AccuSizer FX Nano SIS system is equipped with two sensors, the LE-400; 0.5-100 µm and the FX Nano; 0.15-10 µm (Figure 2). The SIS sampler offers a precise sample volume down to 100 µL with sample recovery.
Figure 2. The AccuSizer FX Nano SIS system Note: showing only the syringe sampler and two sensors, not the pulse height analyzer/counter or computer.
Eight syringes were selected at random from a box of 24. Four were tested using the LE400 sensor without operating the plunger (Figure 3), and four were measured after using the plunger to drive the sample into a clean beaker (Figure 4). The particle size in µm is plotted on the X-axis. The concentration in particles/mL is plotted on the Y-axis.
Figure 3. Without activation, sampled directly from the syringe
Figure 4. With activation, after extracting the sample by pushing the plunger
Results and Discussion
This research was designed in anticipation that activating the plunger would generate a significant increase in particle concentration, which could be attributed directly to the production of extra silicone oil emulsion droplets. In reality, the syringe to syringe difference in reported particle concentration was as good as the difference between activated vs. not activated samples.
As seen in Figure 3, two not activated results have ~10,000 particles/mL >0.5 µm while the other two have ~20,000 particles/mL >0.5 µm. The activated results differ from ~16,000 particles/mL >0.5 µm to 28,000 particles/mL >0.5 µm.
USP 787 was prepared for therapeutic proteins, and while the pass/fail criteria are fixed at 10 and 25 µm matching USP 788, the FDA recommends measuring at smaller particle sizes to identify which formulations will result in more/less aggregation.
The AccuSizer FX Nano SIS system used both sensors to measure the saline from three activated syringes, and the IgG protein in filtered PBS (Figures 5 and 6). The next step was diluting the protein 2:1 with filtered PBS (blue) and saline from the activated syringes as illustrated in Figure 7.
Figure 5. Saline (and silicone oil droplets) from three activated syringes
Figure 6. IgG protein in filtered PBS
Figure 7. IgG diluted with PBS and saline from syringes
The syringe to syringe difference in particle count was best observed at the smaller particle sizes, as illustrated in Figure 5. The AccuSizer FX Nano SIS system was able to spot the smaller particles at 0.2-0.5 µm due to its sensitivity to measure down to 0.15 µm. This same small particle sensitivity is in demand for use in a number of applications. It is preferred in protein analyses, i.e., proteins in filtered PBS, as illustrated in Figure 6, as well as the protein/saline mixture illustrated in Figure 7.
The AccuSizer FX Nano SIS system fulfills all of the prerequisites specified in USP tests 787 and 788. It is a suitable system for sub-visible particle analysis, thanks to its high sensitivity of detecting particles down to 0.15 µm, and low sample volume down to 100 µL.
At this lower range, the sensitivity offers positive outcomes, which can aid researchers conducting therapeutic protein aggregation studies in parenteral drugs and injectables. The AccuSizer FX Nano SIS has additional capabilities over other liquid particle analyzers due to the integration of an innovative technology. Besides being a liquid particle counter, the AccuSizer FX Nano SIS can serve as a highly advanced particle size analyzer.
This information has been sourced, reviewed and adapted from materials provided by Entegris
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