Maura C. Kibbey is director of science and standards, USP Global Biologics.

Maura C. Kibbey, PhD

	Particulates have become a major quality and safety issues, especially in 

Control and Determination of Visible and Sub-visible Particulate Matter in Biologics

was held at The United States Pharmacopeial Convention (USP) headquarters in Rockville, Maryland, on June 26-27, 2017. The first USP workshop to focus specifically on particulates in biologic drugs, the program addressed many issues and challenges unique to biologics-including protein aggregation and immunogenicity-as they relate to particulate control and determination.

	The workshop's goal was to provide a forum for stakeholders to come together, share information, and discuss key issues. This article summarizes highlights from the event. 

	Particulate matter in biologic drugs comes in many sizes, shapes, and materials. Generally, particles may be categorized as intrinsic, extrinsic, or inherent. Intrinsic particles are generated within the manufacturing process and may include silicone oil, rubber, glass, or stainless steel. Extrinsic particles come from outside the process and could include metal, human hair and skin, or dust.

	Inherent particles, such as protein aggregates, are naturally present in protein therapeutics and may be acceptable with the appropriate control strategy. However, just because the particles are inherent does not mean they should be ignored or assumed to be safe. The safety aspects of inherent particles should be assessed throughout a product’s life cycle. Overall, it is important to distinguish between inherent particles and intrinsic or extrinsic particles

	Another way to conceptualize particulate matter is to divide it into two categories: visible and subvisible. Detection of visible particulates (greater than 100–150 microns in diameter) depends on multiple factors such as the lighting, magnification, and container characteristics, as well as the technician’s training and visual acuity.

	Subvisible particles are usually defined as particles that are 1–100 microns in size. In the area of subvisible particulates, there is increased focus on distinguishing between silicone oil and non-silicone oil particles.

	Flow imaging can be used to visualize and count subvisible particles, which involves determining the aspect ratio to classify them. However, aspect ratio is not always reliable and it frequently misclassifies particles. Thus, a method is needed that will recognize patterns in the images.

	Some companies are investing in the use of artificial intelligence to analyze thousands of particle images and categorize them. These algorithms, and equipment using them, scan images from a training set and “learn” to distinguish between the various particle types.

	During the visual inspection process, when a particle is observed in a product, that unit is pulled and either discarded or set aside for further analysis. Several presenters at the workshop emphasized that it is important to have standard operating procedures (SOPs) in place for defects that have been identified during inspection, along with alert and action levels. Taking time to analyze rejected units can reveal clues that will allow manufacturers to prevent similar problems from happening in the future. More importantly, the same problem may be occurring in other lots or even other products, and the analysis may help improve these other materials too.

	Particulates in drug products can form at any stage in the manufacturing process or during storage. Various environmental stress conditions (heat, shock, or vibration) can lead to particle formation. Thus, when developing or refining a formulation, stress conditions should be applied to learn about the formulation’s propensity to form particulates.

	Presenters suggested that, in order to optimize the process and minimize particulate formation, it is best to follow a continuous improvement approach. Continuous improvement can be applied to formulation development, component compatibility, manufacturing procedures, and other processes.  All personnel involved should align on best practices and should also share accountability for the end product.

	Protein aggregation in biologics can compromise the safety and efficacy of the product, noted an FDA presenter. Many triggers for aggregation have been identified, including oxidation, mechanical stress, impurities, pH shifts, and temperature changes. 

	With blood products, another concern is platelet-derived extracellular vesicles (PEVs), which have a 50- to 100-fold higher pro-coagulant activity than resting platelets. Release of PEVs can be associated with the donor’s lifestyle, the collection procedure, and storage conditions. PEVs have a broad size distribution and are associated with adverse events including transfusion-related acute lung injury (TRALI). 

	The gold standard methods for detecting protein aggregates and PEVs in biologics are flow cytometry and light obscuration; however, there is no regulatory guidance available on PEVs. It is always important to use orthogonal methods because these processes are very complex, and each method has limitations, according to the presenter.

	Particles can have adverse effects on the drug product and/or the patient who ultimately receives it. The focus should be on the patient, presenters agreed. For example, if a relatively large number of subvisible particles is detected, one should characterize them and perform a clinical risk assessment. This assessment can reveal information about the impact of particles on the drug product’s clinical performance.

	Important clinical factors to consider include the route of administration, which is often intravenous, and the intended patient population. The most vulnerable patient populations include neonates, young children, seriously ill patients, and the elderly. Protein aggregates and other particulate matter can be immunogenic and can also alter the drug’s efficacy.

	In the worst-case scenario, the patient may develop an autoimmune response. This reaction is sometimes missed because the patient’s illness can mask the adverse effects of the therapeutic product.  It is unclear whether all aggregate types are potentially immunogenic, but there is growing evidence that the size of the aggregates has an effect. For example, vaccine components  are purposely aggregated to increase the immune response to a vaccine.

	FDA speakers recommended that orthogonal techniques based on different separation and detection principles should be used to both enumerate and characterize particles. Presenters from the National Institute of Standards & Technology (NIST) described a standard they developed that is optically similar to protein particles. It mimics the fibrous nature of proteins and is made of abraded ethylene tetrafluoride (ETFE). Unlike particle sets for instrumentation that are spherical and do not resemble proteinaceous material, the ETFE standard is made of a more stable material than protein and also can be easily counted. The NIST study showed that abraded ETFE looked more like protein than did polystyrene beads.

	Another concern raised was that during the final steps before a biologic drug is administered to a patient, particles can form if the drug is mishandled. Thus, it is critical to provide clear, explicit instructions on the administration of drug products. The instructions should describe how these drugs must be handled to protect their potency and efficacy.

	One of the most important and challenging questions raised at the workshop was what is acceptable-how many particles are allowed?  There is no simple answer, said an FDA presenter, adding that FDA looks at the whole profile and cannot specify a limit that will be appropriate for every product. FDA’s evaluation is product specific and is done on a case-by-case basis.

	However, FDA does tell sponsors to collect and provide as much information as possible about their product.  In general, there is no clear evidence regarding a threshold of criticality or cutoff point. How does one justify that a specific level of exposure to particulates is acceptable? In each case, it depends on the product and the patient.

	Assessing drug products for particulates can be challenging for a variety of reasons. Often, reference to the “safety” of a product is connected to particulates, but every product has a risk–benefit profile. To further complicate matters, different patients react differently to the same product.

	One might assume that visible particulates pose a greater risk than subvisible ones, particularly with injectable medications, simply because the visible ones are larger. However, visible particles typically do not fit through the lumen of a needle and thus they do not get injected. For example, 100 microns is about the size of a 36-gauge needle. In contrast, the subvisible particles are very likely to be injected and thus could pose a greater risk.

	In some clinical settings, in-line filters are used to catch any remaining particulates during infusion of the drug to the patient. This approach may sound promising as a final measure for protecting the patient, but in-line filters can be problematic because they can produce particles as well. In addition, hospital personnel might use incompatible filters, unless the appropriate filter is supplied with the medication. Hospitals generally take the position that the drug manufacturer needs to qualify and supply the filter if a filter should be used.

	Some presenters said that in-line filters should only be used if there is a sound, clinically based reason to do so. In some cases, the use of a filter is the only option for a medically necessary drug, where no alternatives are available. However, the in-line filter for a product needs to be qualified as capable of reducing visible and subvisible particles.

	A related issue is the potential for particulates to enter the drug product from the administration set, which is not a filter but rather the standard equipment used to infuse intravenous medications. Administration sets may be loaded with particles, negating the impact of any and all controls and tests designed to prevent particulate contamination.

	If a patient does receive particulates, this has potential clinical implications. In many cases, there is no clinically discernable effect, but this does not mean that no effect has occurred. Particulates can be vaso-occlusive, blocking pulmonary capillaries, or they can act as irritants, leading to soft-tissue granulomas. The composition of the product in terms of organic and inorganic leachates can also be affected.

	When evaluating the intended patient population for a new medication, the factors to consider may include: patient age, comorbid conditions, presence of ischemic tissue, altered immune status, and volume of drug product to be given over a certain time period. If any adverse events occur, finding the root cause and linking clinical effects with particle counts can be very challenging.

	Cell therapy products differ from other types of drug products in several important ways.They may be grouped into the following categories:

		Minimally manipulated-not requiring a biologics license application (BLA)

		More than minimally manipulated-requiring a BLA

		Autologous-coming from and going back to the same patient; because the amount available is limited, destructive testing cannot be done

		Allogenic-coming from a single source and, typically, going to many patients.

	During manufacturing, cell therapy products cannot be purified or filtered using the standard approaches to remove particulate matter from drug products. In addition, these products are often opaque rather than clear, limiting the effectiveness of subvisible and visual particle detection methods. 

	Differentiating between contaminants and the product itself, which typically contains particulates too, is often complicated, noted one of the FDA attendees. There are many challenges involved in monitoring particulate matter, therefore emphasis should be placed on limiting introduction of particulate matter that may be in raw materials or introduced by the manufacturing process.

	Particulates that affect cell therapy and gene therapy products do not come from one single source. Instead, particulates are generated by many sources, often with no consistency. Particles can come from the original biological components, such as solid tissue or bone marrow. Other sources are tubing, bags, vials, and stoppers that come in contact with the materials and could also contain and release particulates. Other sources for particulates include the environment, personnel, instruments, and excipients.

	Although FDA recommends using orthogonal methods to evaluate and analyze biologic drug products, this is not always possible. In some cases, there is no orthogonal method available. In the area of cell therapy, there are no established industry-specific standards for particles.

	In one presentation about cell therapy and gene therapy, the speaker emphasized the growing challenge of controlling particulates in cell/gene therapy products, and the need for new detection methods. More scientists and their companies are starting to admit that they are encountering problems and challenges with particulate control and determination. However, there is still a lack of data on this issue, and manufacturers are searching for solutions to various problems.

	Control of particulates is as important as developing methods to reduce them. FDA presenters advised cell therapy manufacturers to first determine the number, size, and type of particles, and then try to control and/or reduce particles. The next step is identifying the likely contributing sources, then developing mitigation strategies such as:

		switching to better-quality source material

		evaluating and improving cleaning practices

		finding ways to reduce the number of handling steps

Inspecting products for visible particles

	As one member of USP’s visual inspection expert panel mentioned, before 2010, there were few standards for performing particulates inspection. Now, it appears, people are taking some consistent and similar approaches.

	Today, performing a robust 100% inspection of the finished drug product is a must, according to members of the expert panel. The goal is to use a statistical approach to inspection to achieve a product that is essentially free of visible particulates. The concepts involved were defined in 

USP general chapter <790> “Visible Particulates in Injections.” 

	However, striving for zero defects seems unrealistic, according to workshop attendees, because visual inspection is such a probabilistic process.

	Another dilemma is knowing when to stop looking for particulates. USP inspection conditions have been defined and are now harmonized with the 

; they apply to extrinsic and intrinsic particles. In fact, as a number of workshop participants noted, the three major pharmacopeias are now 

(i.e., USP chapter <790> versus EP 2.9.20 and JP 6.06), which is a big step forward.

	Another aspect of inspection is the “difficult to inspect parenterals (DIPs).” A product may be a DIP due to the container type, even if the solution itself is clear. With DIPs, there is a diminished capacity for 100% visual inspection; the actual rate is more like 70%–80%, according to a presenter on this topic.  Stakeholders need to develop and use testing and controls that can detect particles and statistically significant changes throughout the lifecycle of a product.

Themes and concepts discussed in the workshop

		Particle control involves many factors and should be characterized and monitored throughout the product lifecycle.

		Even though FDA does not recommend specific orthogonal methods to detect particles, the consensus is that they are important, as is quality by design (QbD), which requires evaluating each step in a procedure.

		More rigorous particle characterization is necessary. In particular, silicone oil poses many concerns because it can interact with products and be immunogenic.

	Based on the issues raised during this workshop, USP will consider suitable revisions to the existing chapters or the creation of new standards for certain classes of biologics. 

Articles

The workshop focused on topics specific to biologics, including particulate control and determination, becoming the first workshop of its kind for the organization.

USP Workshop Focuses on Biologics

As pharmaceutical products have evolved over the decades, so have processes for their manufacture and analytical techniques for understanding their composition. For every medicine taken by a patient, it is important for a drug maker to be able to identify impurities of any significance that can result from its manufacturing process and to take measures to control for unwanted impurities, especially because they may affect the safety and efficacy of a product.  

An impurity is a substance occurring in a finished drug product that plays no functional role and is not an inherent component of the pure form of the intentionally used ingredients. It can be the result of a manufacturing process, can be added intentionally as a catalyst during drug synthesis, or can be a byproduct of degradation that emerges during the lifecycle of a drug.  

Beginning in the 1990s, the International Conference on Harmonization (ICH) began the development of guidelines relating to impurities in pharmaceuticals. ICH guidelines provide a framework for regulatory filings for new drugs. This body of work on impurities led to what is now well-recognized as the ICH Q3 series, which includes guidelines (allowable levels) for organic and inorganic impurities as well as residual solvents. In developing its own standards for impurities, the US Pharmacopeial Convention (USP) has worked to harmonize with ICH, where feasible, while maintaining its goals of modernizing and creating standards that reflect current-day practices in industry that help to ensure drug quality. 

A nonprofit scientific organization, USP develops standards for the identity, strength, quality, and purity of medicines and their ingredients. In addition, USP's standards include those for specific impurities associated with drug substances and drug products. Documentary standards in USP's compendia—

)—are published as individual monographs and general chapters as well as 

With regard to organic impurities, USP established in recent years standards for residual solvents in drug products, drug substances, and excipients. In addition, USP has initiated a review of other general chapters relevant to organic impurities overall and has developed revised standards for elemental impurities. In 2013, USP will host a workshop on yet another group of impurities—residual host-cell proteins and DNA in biotechnology products.  

Organic impurities cover a wide spectrum of compounds that have varying structures, behavior, and characteristics. They can result from process manufacturing, storage conditions, or even degradation. Some may be volatile and may be very liable to change as a result of exposure to heat, light, or other external factors.  

A USP Expert Panel has been charged with making recommendations on revisions to General Chapter <1086> 

Impurities in Drugs Substances and Drug Products

Ordinary Impurities and General Notices 5.60

. Composition of the advisory Expert Panel includes representatives from pharmaceutical and over-the-counter (OTC) product manufacturers and their trade groups (i.e., the Consumer Healthcare Products Association, Pharmaceutical Research and Manufacturers of America, and others), members of USP's Expert Committees on Physical Analysis, Small Molecules and Toxicology, and liaisons from FDA.  

General Chapter <1086> includes some key definitions associated with impurities that are aligned with those established by ICH, other pharmacopoeias and current FDA guidance. General Chapter <466> applies a relatively simple, yet old, method—thin-layer chromatography (TLC)—for impurity detection. Although TLC enables a user to compare a test article against a known standard and confirm the presence of that article in a sample, it is limited in its ability to distinguish between APIs, excipients, or impurities and to accurately quantify impurities within a given sample. 

The Expert Panel's review will take into account organic impurities associated with hundreds of monographs appearing in the 

, which include OTC drugs and various dosage forms associated with a single OTC. As part of its work, the Expert Panel is assessing the feasibility of categorizing different types of organic impurities (e.g., heterocyclic compounds) and, based on those categorizations, will explore relevant methods for detection and measurement that may be applied to multiple monographs within a single group.  

Although traditional small-molecule drugs are made through chemical synthetic routes, many biologic and biotechnology products are made using recombinant technologies in host cells designed to produce a genetically engineered therapeutic protein. These recombinant products are often transcribed from human DNA sequences that are placed into host cells such as mouse cell lines, yeast cells, or 

 After the cells produce the proteins, they are purified and characterized. Manufacturers of these protein products must be able to demonstrate that their final products contain very low levels of host-cell DNA and proteins because there could be a risk of tumorigenicity or immunogenicity, respectively, from these materials when given to the patient. Currently, USP is developing a chapter on residual DNA, and at least two material reference standards will be available to support the chapter. USP also is developing a general information chapter containing best practices for critical reagent development and characterization as well as development, validation, and use of host-cell protein measurement procedures. 

In June 2013, USP will cohost a workshop with the BioPharmaceutical Emerging Best Practices Association (BEBPA) focused on the measurement of residual DNA and host-cell proteins in biotechnology products. The meeting will feature current industry practices and provide perspectives from the regulatory community. For a workshop description and registration information, see 

Regarding elemental impurities, USP has developed two relevant general chapters—<232> 

General Chapter <232> specifies limits for selected elemental impurities, including limits for mercury, cadmium, arsenic, and lead—toxic elements commonly found in the environment. In General Chapter <233>, procedures for identifying impurities using inductively couple plasma technology are described. In addition, General Chapter <233> provides validation criteria should a manufacturer choose to use procedures other than the one described in the new general chapters. As is the case with residual solvents, the elemental impurities chapters will be applied to articles recognized in 

provision, expected to be proposed in January 2013 in 

 and become official May 1, 2014. For updated information about the current status of USP's standards on elemental impurities, see 

is senior scientific liaison, all with the US Pharmacopeial Convention (USP). 

USP's focus in 2013 involves standards relating to organic impurities, measurement of residual DNA and host-cell proteins in biotechnology products, and elemental impurities.