Assessing Therapeutic Protein Injections

The new USP <787> Subvisible Particulate Matter in Therapeutic Protein Injections test, which is very similar to the USP <788> for parenteral drugs, deals with therapeutic protein injections and makes changes for smaller test aliquots and smaller test product volumes. This article describes the sample test using the new USP <787> procedure. The same standardization steps as outlined in the USP <1788> are followed:

  • Volume accuracy
  • Flow rate
  • Sensor resolution
  • Calibration
  • Count accuracy

It is recommended to perform the following system control tests:

  • Blank test - Five aliquots of degassed particle-free water are measured, and the count should be less than 1 particle/mL > 10 µm
  • System suitability verification - The USP particle count reference standard is analyzed, and the count limits are passed

Sample Preparation

Individual units can be tested if there is an adequate volume. If the volume is not sufficient, the units are mixed, and the contents are combined to obtain the desired volume, typically in the range of 0.2 - 5.0 mL. The sample is then degassed and gently mixed again.

Analysis

Four aliquots are analyzed. The particles within the size range of interest that include particles >10 and 25 µm are then counted. The first result is ignored, and the next three results are then averaged.

Pass/Fail Criteria

  • If the container volume < 100 mL; less than 6000 particles/container > 10 µm and 600 particles/container > 25 µm
  • If the container volume > 100 mL; less than 25 particles/mL > 10 µm and 3 particles/mL > 25 µm. In addition, per container limits are not exceeded for the < 100 mL criteria above.

Most of the system preparation and measurement steps are very similar to the USP <788>. These tests can be efficiently carried out using the standard Entegris AccuSizer SIS system (Figure 1). The ability to handle sample volumes as low as 0.2 mL (200 µL) is the major difference. Samples can be accurately measured down to 250 µL by the SIS sampler, making the system an ideal tool for analysis of small volume protein samples.

Single Sensor AccuSizer SIS

Figure 1. Single Sensor AccuSizer SIS

FDA Guidance on Protein Injections

The Food and Drug Administration (FDA) released a Guidance for Industry document, "Immunogenicity Assessment for Therapeutic Protein Products" in August 2014. In this document, the FDA states that, “it has been recognized that subvisible particulates in the size range of 0.1–10 microns have a strong potential to be immunogenic, but are not precisely monitored by currently employed technologies… As more methods become available, sponsors should strive to characterize particles in smaller (0.1–2 microns) size ranges.”

In addition to the FDA recommendation, the common understanding that studying protein aggregation phenomenon needs measuring the particle size less than 10 and 25 µm required for the USP <787> testing also drives the desire to perform measurements at these smaller sizes.

AccuSizer FX Nano SIS System

The two sensor AccuSizer FX Nano SIS system (Figure 2) enables users to easily and accurately carry out particle count measurements down to 0.15 µm (150 nm). The two sensor configuration integrates the FX Nano sensor into the LE-400 sensor employed in the single sensor system. The same syringe sampler is employed in conjunction with two pulse height analyzers (counters). Separate research software helps to achieve sensitivity down to 0.15 µm by integrating the two results. In addition to the small sample volume and recovery features, the two sensor AccuSizer FX Nano SIS system has been proven to be the ideal system for conducting protein aggregation studies.

Two sensor AccuSizer FX Nano SIS

Figure 2. Two sensor AccuSizer FX Nano SIS

Results

The procedure was started by preparing immunoglobulin G (IgG), ~150 kDa, 1% in filtered PBS, followed by measuring the sample in an undiluted condition with the AccuSizer FX-Nano SIS two sensor system. The next step was to pass the protein sample through a 0.2 µm filter and analyze it again using the AccuSizer FX-Nano SIS system. Figure 3 shows the results before and after filtration. The results for the PBS are also presented in black. After the filtration step, the concentration was lowered from 9.7 to 3.1 x 108 particles/mL. The reduction in the tail of aggregated particles can be clearly seen and easily determined by the AccuSizer FX-Nano SIS system.

IGG before (blue) and after (red) filtration

Figure 3. IGG before (blue) and after (red) filtration

This information has been sourced, reviewed and adapted from materials provided by Entegris

For more information on this source, please visit Entegris

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