Melanie Zerulla-Wernitz is team leader project & service analytics,Vetter Pharma-Fertigung GmbH & Co. KG.

Melanie Zerulla-Wernitz

The development of complex biological drug substances such as monoclonal antibodies, interferons, peptides, vaccines, and ophthalmics is boosting demand for innovative delivery mechanisms (1). Many drug manufacturers are, therefore, opting for prefilled drug-delivery systems to administer new therapeutics. 

	However, employing prefilled delivery solutions can give rise to specific challenges. Particles can appear for a variety of reasons, during the manufacture, storage, or transportation of prefilled products. In turn, these particles can have an impact on the drug’s effectiveness. For example, if the particles are composed of large protein aggregates, the patient’s cells will only be able to partially absorb the drug or, for some products, will not be able to absorb them at all. Furthermore, particles can trigger unwanted side effects in the patient, such as autoimmune responses. 

	For the aforementioned reasons, authorities have-over the past few years-been steadily tightening up regulatory demands in regards to sub-visible and visible particles. Today, the authorities expect bio/pharma companies to develop, validate, and establish manufacturing processes, storage conditions, and transport operations in a manner that will eliminate the cause for the appearance of particles, as well as to ensure that particles and other contaminants are kept at a minimum. 

	Yet, for companies to be able to comply with the authorities’ expectations, deep knowledge of the processes involved is required. Additionally, identification and characterization of the particles are becoming ever-more significant. 

	The development of innovative analytical techniques has given rise to new investigative possibilities, in particular for sub-visible particles, which enable pharma and biotech companies to implement robust, high-quality processes for the manufacture of drug products in prefilled drug-delivery systems.

	There are different types of particles and different reasons why they are generated. Biologics are particularly prone to producing particulate matter.

	Extrinsic particles are foreign, additive particles and not part of the drug product formulation or primary packaging material. Extrinsic particles can result from the environment, such as cellulose fibers originating from disinfectant cloths, a human user, rubber, plastic, or metals. Because these particles arise externally, they may not be sterile and could be considered contaminants for the filled unit.

	Intrinsic particles are generated directly from the formulation of the drug product or during the manufacture of the drug product. Intrinsic particles such as protein aggregates may be the result of inherent properties of the drug product, interactions of the drug product formulation components or their contact with primary packing materials or processing aids. They may also emanate from the active drug substance.

	Inherent particles are generated from the formulation itself, for example, when an active substance or an excipient forms a haze or aggregates. This particle formation can be due to greater shearing forces, among other causes. Inherent particles do not affect the effectiveness of the drug and, therefore, do not have a negative impact on the patient.

	Air bubbles (as well as silicone oil droplets) are not particles per se, but almost all methods of analysis will reveal them as such because they are highly reflective. Air bubbles are most often generated by sample preparation.

	Silicone oil droplets specifically occur in syringes and cartridges, which, in contrast to vials, must be siliconized to enable the stoppers to glide smoothly in the glass barrels. Drops of silicone oil cannot be entirely avoided but they can be minimized. The amount of silicone oil applied can be reduced but only to the point that it does not impede break-loose and glide forces. This is a particular challenge for ophthalmic drugs.

	Particles also come in a variety of sizes. There are visible particles (approximately 100–150 µm and larger), which can be spotted with the naked eye during visual inspection and without any outside assistance such as magnifying glasses or microscopes. Conversely, subâvisible particles are in the nanometer to micrometer range. These particles include drug substance aggregates, silicone oil drops, fibers, and other materials. For injectables, the 

) defines, in chapter <788>, limit values for sub-visible particles equal to, or greater than, 10 μm (N = 6000) and 25 μm (N = 600) (2). The limit values for ophthalmic drugs are even more strict, namely equal to, or greater than, 10 µm (N = 50) and 25 µm (N = 1), see 

	Regulatory authorities, such as FDA, have tightened their requirements for the identification and characterization of particles over the past years, see 

<790> and <1790> (4,5). The authorities now offer a level of orientation when it comes to the visual inspection of filled units with regard to visible particles in injectable drugs. In addition, the sections of the corresponding pharmacopeias covering sub-visible particles are regularly reviewed and expanded where needed. The new guidelines require that companies not only discover the particles and sort out the visible particles contained in prefilled drug-delivery systems, but they also have to focus on particle characterization and root-cause analysis of particle type and source in commercial batches. The ultimate goal is to adjust manufacturing processes so that units contaminated with visible particles can be avoided altogether, and sub-visible particles are understood and avoided as much as possible. To understand the origin of particles, a close collaboration between different departments, such as development, manufacturing and quality control, is needed.

	There are a number of analytical approaches used for the determination, characterization, and identification of particles. The standard procedure to determine visible particles is a visual check of the filled units combined with a subjective description of any visible contaminants. Until now, the standard procedure for quantification of sub-visible particles in the micrometer range has been light obscuration. This technique, which is a compendial method for routine testing used in batch release, determines the number and size of sub-visible particles in the 1–100 µm range. However, the technique also has some limitations, especially since a description of the particle’s morphology and chemical characteristics is not possible. 

	Analytical labs are now establishing a range of novel analytical techniques to provide comprehensive particle characterization and identification, in particular, for sub-visible particles. These techniques are used primarily for testing and measuring the limits of the compounding, mixing, and filling procedure design during development. It is also possible to apply these techniques to any issues arising in commercial batches as they are capable of providing additional information that can be used to assist in performing a root-cause analysis. For example, these methods include the following (

Archimedes’ resonant measurement (Malvern Panalytical):

 This technique is used to detect sub-visible particles in the submicron range. Particles between 50 nm and 5 μm are transported through a mechanically resonating microfluidic channel. The mass, dry mass, and size of particles are calculated. The detection of particles with different buoyancy makes it possible to distinguish extremely small sub-visible particles such as a drug protein aggregates from silicone oil droplets.

Micro-flow imaging (MFI, Protein Simple):

 MFI combines the possibilities of digital microscopy with modern microfluidics, producing high-resolution images of sub-visible particles between 1 μm and 70 μm. Image analysis permits the determination of the morphology, intensity, and coincidence of particles. This, in turn, provides information about the particle type (e.g., air bubble, silicone oil droplet, protein aggregate, or fiber). Therefore, by combining information about particle number and size with information about particle shape and transparency, morphological data can be generated that will characterize individual particle subsets by using a modern application software.

 This approach combines the automated static imaging capabilities of a high-resolution, modern digital microscope with the chemical identification of individual particles using Raman spectroscopy. This method is suitable for sub-visible and visible particles because it can be used to classify particles over a very wide range (1 μm–1000 μm) by size and morphology, as well as chemically by means of Raman spectra.

ETAC Proview (Bosch Packaging Technology):

 This computer-based automated inspection system, a digital visual inspection at laboratory scale, is used for research and development, and enables objectification of the manual visual inspection. Photos and videos can be made of filled units from different perspectives using two cameras and a range of lighting options. Application software permits the precise evaluation of visible mobile and immobile particles in each unit. By contrast, the human eye can only count up to a limited number of particles (

	One effective way to gather reliable data when identifying and characterizing particles is for the outsourced service to create a team of experts with comprehensive knowledge of a specific topic (i.e., particulate matter). Each new project will then further expand the formed team’s knowledge. By using innovative analytical equipment the team will be able to perform most of the analytical methods used in the field of particle measurement.  

	Through the use of a specific team of experts and by expanding the team’s knowledge on particular topics, reliance on an additive external laboratory, which requires greater organizational effort and additional sample shipment, may be circumvented as everything can be achieved via a single outsourced service provider. As the specialists come to know the manufacturing processes, they can continuously optimize the analytical services and offer a range of support to help meet the challenges inherent to the topic of particulate matter.

	The number and types of biological drugs in development are constantly growing which, in turn, fosters stricter guidelines from regulatory authorities, especially with regard to particulate matter. Manufacturers will need to adjust their analytical techniques as well as production processes to quickly identify and reduce the causes of particle generation. 

	As a result of the need to adjust techniques and processes, analysis and characterization of particles have become a priority. By drawing on technological advances, the latest methods of analysis, and experienced specialists, bio/pharma companies will not only gain insights into the types and sizes of particles, but will also be able to perform fast and efficient root-cause analysis. 

	Manufacturers of parenteral drugs will benefit in the long-term from detailed data about the processes involved in commercial manufacturing. These manufacturers will also be able to comply with the stricter regulatory guidelines and customer demands while maintaining high quality standards. 

	1. K.J. Wrigley, Pharm. Tech., 41 (10) 32–35 (2017).
	2. USP, 

 General Chapter <788>, “Particulate Matter in Injections” (US Pharmacopeial Convention, Rockville, MD, 2012).
	3. USP, 

 General Chapter <788>, “Particulate Matter in Ophthalmic Solutions” (US Pharmacopeial Convention, Rockville, MD, 2012).
	4. USP, 

 General Chapter <790>, “Visible Particulates in Injections” (US Pharmacopeial Convention, Rockville, MD, 2014).
	5. USP, 

 General Chapter <1790>, “Visual Inspection of Injections” (US Pharmacopeial Convention, Rockville, MD, 2015. 

	When referring to this article, please cite it as M. Maier and M. Zerulla-Wernitz, “Analysis of Sub-Visible Particles," 

is head of the Analytical Science Laboratory, Project & Service Analytics, Vetter Pharma-Fertigung GmbH & Co. KG

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Novel analytical methods may help biologics manufacturers respond to stricter regulations on particulate matter.
