Particulate Matter in Injections by Light Obscuration
Counts sub-visible particles of 10 µm and above and 25 µm and above in an injection using a light obscuration counter, and compares the counts per container (or per mL) with pharmacopoeial limits.
- Light obscuration particle count test
- Liquid particle counter
Reference: Typical approach based on USP <788> method 1 and Ph. Eur. 2.9.19. Always follow the current chapter and your approved STP.
Why this method works
The injection is drawn through the narrow sensor of a light obscuration counter. Each particle blocks part of a light beam, giving a pulse whose height depends on its size. Pulses are counted into size classes calibrated with certified spheres. Several portions are measured, the first is discarded and the rest are averaged. The average count per portion is scaled to particles per container (small-volume injections) or per mL (large-volume injections).
Instruments used
Reagents & standards
| Reagent | Grade / specification |
|---|---|
| Particle-free water | Purified water filtered through a membrane of 1.2 µm or finer |
| Glassware | Cleaned and rinsed with particle-free water |
Preparing the sample
- 01
Blank
Run particle-free water to show the environment and glassware are clean.
- 02
Mix
Invert each container gently about 20 times.
- 03
Pool
For small units, pool enough containers to give at least 25 mL.
- 04
Degas
Let stand or apply gentle vacuum to remove bubbles.
- 05
Measure
Run four portions of not less than 5 mL each.
- 06
Calculate
Discard the first; average the rest and scale per container or per mL.
Procedure
- 1Carry out the test under a laminar air flow cabinet wearing powder-free gloves.
- 2Run a blank of particle-free water (e.g. 5 portions of 5 mL); it should meet the environment limit (commonly not more than 25 particles ≥ 10 µm in 25 mL).
- 3Wipe the outside of each container. Mix by inverting gently about 20 times; do not shake.
- 4For containers of 25 mL or more, test units individually. For smaller units, open and pool the contents of enough units (commonly 10 or more) into a clean vessel to give at least 25 mL.
- 5Let the sample stand, or apply gentle vacuum, until no air bubbles are visible.
- 6Measure four portions of not less than 5 mL each and record counts at ≥ 10 µm and ≥ 25 µm.
- 7Discard the first portion and calculate the mean of the remaining portions.
Standard preparation
- 1No standard is run with each test; sizing calibration, sensor resolution, count accuracy and volume accuracy are verified at the calibration interval.
Notes for analysts
- Most high results come from contamination during handling; repeat the blank before blaming the product.
- Large-volume results are per mL: average count per portion ÷ portion volume.
- Protein products may contain translucent particles that light obscuration undersizes; orthogonal methods (e.g. flow imaging) are used in development.
Method parameters (typical)
| Instrument | Light obscuration particle counter calibrated with certified polystyrene spheres |
|---|---|
| Size channels | ≥ 10 µm and ≥ 25 µm |
| Portions | Four portions of not less than 5 mL; first discarded |
| Environment | Laminar air flow; particle-free water blank |
| Limits (small-volume, ≤ 100 mL) | Typically not more than 6000 particles ≥ 10 µm and 600 particles ≥ 25 µm per container (see current chapter) |
| Limits (large-volume, > 100 mL) | Typically not more than 25 particles/mL ≥ 10 µm and 3 particles/mL ≥ 25 µm (see current chapter) |
System suitability & sequence
| Parameter | Limit |
|---|---|
| Blank (environment) | Typically not more than 25 particles ≥ 10 µm in 25 mL of particle-free water |
| Instrument calibration | Sizing, resolution, count accuracy and volume accuracy within due date |
Typical sequence
- 1Particle-free water blank
- 2Sample pool / unit: portion 1 (discard)
- 3Portions 2–4 (average)
- 4Next sample
Calculation with a worked example
Where
- A
- Mean particle count of the portions analysed (portions 2–4)
- VT
- Total volume of the pooled sample (mL)
- VA
- Volume of each portion analysed (mL)
- n
- Number of containers pooled
Given
- Product
- 2 mL ampoules; 15 ampoules pooled
- VT
- 30 mL
- VA
- 5 mL
- n
- 15
- Counts ≥ 10 µm (portions 2–4)
- 183, 171, 177
- Counts ≥ 25 µm (portions 2–4)
- 6, 4, 5
Working
- ≥ 10 µm: A = (183 + 171 + 177) ÷ 3 = 177 per 5 mL
- Per container = (177 × 30) ÷ (5 × 15) = 5310 ÷ 75 = 70.8
- ≥ 25 µm: A = (6 + 4 + 5) ÷ 3 = 5 per 5 mL
- Per container = (5 × 30) ÷ (5 × 15) = 150 ÷ 75 = 2.0
≈ 71 particles ≥ 10 µm and 2 particles ≥ 25 µm per container, well within the typical small-volume limits of 6000 and 600 per container: complies.
What result passes
- Small-volume injections (≤ 100 mL): typically not more than 6000 particles ≥ 10 µm and not more than 600 particles ≥ 25 µm per container.
- Large-volume injections (> 100 mL): typically not more than 25 particles per mL ≥ 10 µm and not more than 3 particles per mL ≥ 25 µm.
- If the light obscuration test fails, USP <788> allows testing by the microscopic particle count method (method 2). Always check the current chapter.
Validation parameters (ICH Q2(R2))
| Parameter | Typical requirement |
|---|---|
| Suitability | Product does not interfere (colour, viscosity, bubbles, precipitation); otherwise dilute or use the microscopic method |
| Precision | Repeat counts on the same pool agree |
| Robustness | Mixing, degassing and pooling steps shown not to add or remove particles |
Run this method in LimsPulse: specifications and STPs under version control, results calculated and checked against limits, and a signed Certificate of Analysis.