Evaluation and Identification of

Subvisible Particulate Matter in

Injections – JUNE 2022
The presence of particulate matter (PM) in parenteral drug
products is a well-known challenge for pharmaceutical
companies due to the potential quality and safety risks involved.
In biotherapeutics, critical attributes related to efficacy,
potency, clinical safety, and immunogenicity can be affected by
PM.1-3 
PM is defined as extraneous mobile undissolved particles
(excluding gas bubbles) that are unintentionally present in
solutions.4
Several USP chapters describe the measurement of PM, namely:

  <787> Subvisible Particulate Matter in Therapeutic Protein Injections

 <788> Particulate Matter in Injections
 <789> Particulate Matter in Ophthalmic Solutions
 <790> Visible Particulates in Injections

  
In this article, the scopes of <787> and <788> will be discussed since both chapters
address injections and have the same regulatory requirements according to their
nominal volumes for subvisible PM.

As compared to visible PM, subvisible PM is unnoticeable without a microscope and is

harder to detect. Subvisible particulates present a greater challenge for detection and
higher risks that they might go undetected due to their smaller size. As a result,
establishing PM profiles for subvisible particulates is highly demanding, and made more
so by instrumental limitations and sample handling challenges (to be discussed further
in the article). 

The occurrence of subvisible PM can stem from several different sources. Particularly,
the subvisible particulates found in sterile drug products originating from packaging
components and delivery systems are a major concern for drug manufacturers, with
potential sources attributed to material components, process aids used to assemble the
system, secondary packaging, insufficient washing, and contamination during fill-finish
process.5 Therefore, understanding the method of determination and enumeration are
critical.

What are Subvisible Particulates and Their Sources?

Subvisible particulates are particles < 100 µm in size and are not visible to the naked
eye.6 A related chapter in USP <1787> Measurement of subvisible particulate matter in
therapeutic protein injections categorizes this undesired PM as intrinsic, extrinsic, or
inherent. 

Intrinsic PM arises from sources related to formulation ingredients, packaging,

manufacturing processes and equipment.  This may also stem from the poor selection
or cleaning of components and systems. An example is silicone oil, which is an
important manufacturing and product constituent that may ultimately affect PM count. 

Environmental factors, that is, materials that are not part of the formulation, package, or
assembly process, are the main sources of extrinsic PM. The production environment,
including personnel gowning and behaviours can contribute extrinsic PM to the filled
product. 

Additionally, some products contain a small level of unintended PM that is inherent to

the product and therefore, are considered potentially acceptable characteristics of the
product. Proteinaceous aggregates are examples of inherent PM found in therapeutic
protein drugs, formed by interactions of the protein with itself or with other formulation
ingredients. Heterogeneous particulates consisting of more than one chemical entity
may also result from protein aggregation. They can be classified as extrinsic or intrinsic
based on the nonproteinaceous component that the protein combines with to form the
aggregate.  

Intrinsic and extrinsic particulates account for the majority of the PM originating from
elastomeric closure components and their manufacturing process. Different types of PM
have diverse effects on safety and product stability. It is critical to perform PM
evaluations and concentration measurements to understand the nature of the
particulates involved. Thereafter, the amount of PM can then be minimized in the final
drug product upon PM identification and ascertaining their sources. 

Determination of PM

USP <787> and <788> describe two procedures for measuring subvisible PM in
parenteral products by direct testing of the drug product itself: light obscuration (LO)
particle count test and microscopic particle count test. 

USP <1788> suggests the use of Flow Imaging (FI) method, not only to complement the
above methods, but also allows particles to be characterized into categories of intrinsic,
extrinsic and inherent, in the case of biologics for the purpose of risk assessment and
continuous improvement. For the simplicity of this article, FI will not be discussed. 

LO Particle Count Test vs Microscopic Particle Count Test

An LO particle count test is based on the principle of light scattering that enables
automatic determination of particles size and the number of particles according to size.
A laser beam that is shone through the liquid sample as it is drawn though an optical
flow cell, will be scattered or absorbed by any particles, air bubbles or liquid droplets,
thus reducing the total transmitted light. The scattering pattern or “shadow” produced
on the light-sensitive detector can then be translated to information on particle size and
quantity.

A microscopic particle count test is an alternative technique to LO for subvisible particle

analysis. It involves the use of a suitable binocular microscope, a filter assembly for the
retention of particulate matter, and a membrane filter for examination. The liquid
sample is filtered and inspected through the microscope, followed by manual counting
of particles above the size threshold. However, liquid droplets and aggregated proteins
that can pass through or absorb onto the filter are not reliably counted by this method. 

As a fully automated method, LO can analyze large volumes of samples quickly and
easily, with high resolution, minimal operator errors, and without requiring
interpretation of data.  Therefore, LO is generally the preferred method and the
microscopic particle count test is applied only when the former is not applicable. For
example, drug products (i.e., emulsions, colloids and liposomal preparations) with high
viscosity and/or high opacity are not suitable for LO method. Products that generate air
or gas bubbles upon drawing into the sensor are also more appropriate for microscopic
particle count testing. This is because these bubbles might be detected as a particle,
resulting in false positive data.  

As for the microscopic particle count test, complete sample measurement is performed
by filtering through the entire sample. The PM retained on the membrane can be used
for characterization and identification, if desired (LO method does not allow sample to
be reused for further characterization after the count test has been conducted).
However, this process  requires analyst expertise and experience. 

Due to the inherent limitations in particle count techniques, it may be beneficial to

utilize both test methods to accurately quantify the number of particles present.
  

Regulatory Requirements according to USP <787> and <788>

With the different test methods there are different sets of specifications for parenteral
infusion or solutions for injection supplied in containers according to volume. The
acceptance criteria are illustrated in Table 1 based on each method and the nominal
volume of contents within the containers supplied.

West as a Scientific Destination

Understanding of the relevant particle count technology, method capability, and the
particle source is critical in mitigating the risks associated with subvisible PM. To achieve
accurate and reliable data on subvisible particle count, West can provide comprehensive
guidance on the use of analytical methods described in USP <787> and <788>, as well as
additional particle characterization methods described in USP <1787>  by orthogonal
techniques. West’s Analytical Laboratory is capable of performing characterization and
identification of particles and this can be performed as part of the development phase,
root cause analysis for nonconformance investigations, stability study, and risk
assessment. 

Information and data obtained from these testing methodologies will aid in the selection
of proper components suitable for each application. West is equipped with the
appropriate experience, knowledge, and facilities to perform evaluations according to
the standards cited and offers testing through its Integrated Solution platform. `

References

1. Walpot H, Frank RP, Burchard WG, Agternkamp C, Muller FG, Mittermayer CKG.
Particulate contamination of intravenous solutions and drug additives during long- term
intensive care. Anaesthesist. 1989;38:617-621.
2. Langille SE. Particulate matter in injectable drug products. PDA J Pharm Sci Technol.
2013;67(3):186-200.
 3. Bukofzer S, Ayres J, Chavez A, et al. Industry perspective on the medical risk of visible
particles in injectable drug products. PDA J Pharm Sci Technol. 2015;69(1):123-139.
4. USP <788> Particulate Matter in Injections. Rockville, MD: US Pharmacopeia.
5. Rech J, Fradkin A, Krueger A, et al. Evaluation of particle techniques for the
characterization of subvisible particles from elastomeric closure components. J Pharm Sci.
2020;109:1725-1735.
6. USP <1788> Methods for Detection of Particulate Matter in Injections and Ophthalmic
Solutions. Rockville, MD: US Pharmacopeia.