A very particular problem

Lives are on the line when it comes to correct understanding of how therapeutic proteins can form particulates says Professor John Carpenter.

The positive impacts of therapeutic proteins on the lives of people suffering from cancer, diabetes, multiple sclerosis, Crohn’s disease, ALS, lupus, arthritis and other diseases and conditions have attracted significant investment in drug research and development.

As a result, hundreds of therapeutic protein-based drugs have earned FDA approval, with enormous benefits to human health. With the rapid growth and acceptance of these biologics, more and more information about impacts of product quality on patient outcomes has become available. Based on this growing body of evidence, one of the most important product quality attributes is the concentration and sizes of subvisible particles.

Protein particles can form in the drug formulation at every step in the manufacturing process and throughout the supply chain, including while sitting in storage and at the point of injection. Similarly, particles of foreign materials can enter the formulation at steps throughout a products’ life history. Sources of foreign particles include processing equipment such as filling pumps, container-closures and delivery systems such as bags of intravenous saline. The materials include rubber, glass, stainless steel, plastic and silicone oil. Proteins can adsorb to foreign particles creating hybrid particles, as well as potentially stimulating further protein aggregation.

Critical insights into the causes of product quality problems have been obtained by using the modern methods for characterisation of nano- and microparticles

Homogenous protein particles, foreign particles and hybrid particles can reduce product quality and potentially induce adverse effects in patients. Such adverse effects include infusion reactions and immunogenicity. For example, some patients respond well to therapeutic protein treatments only to experience a sudden decline in response as their symptoms return due to the immune system’s response to the protein drug. In some documented cases, this enhanced immunogenicity has been causally linked to the presence of protein aggregates and/or to micro- and nano-particles in the formulation. Regulators require drug manufacturers to test for particulate matter, determine the sources of particles and develop control strategies for particles.

The problem with particles… Mitigating particulate formation and contamination requires a detailed understanding of how a given protein aggregates and of how the various types of bioprocess equipment and drug containers and delivery systems shed particles during production and drug administration. Gaining these critical insights depends directly on the capabilities of analytical instruments to quantify and characterise both nano- and microparticles.

For example, employing these analytical methods in one recent study documented that nano- and microparticles are present in intravenous saline and that proteins delivered in that saline via an intravenous administration system form additional particles. Importantly, it was observed that commonly used in-line filters were effective at removing microparticles, but not nanoparticles.¹ Another study showed that positive displacement filling pumps shed stainless steel nanoparticles onto which antibody molecules adsorbed, resulting in pumping-induced micropartices.² And several studies have shown that silicone oil from prefilled syringes can cause protein aggregation and particle formation.

In general, even routine handling (or mishandling), such as agitation and exposure to light, can trigger aggregate and particle formation. For example, recent unpublished studies show that in some hospitals, protein drugs in IV bags or syringes are transported via a pneumatic tube system. The mechanical shock occurring when the transport container hits the receptacle wall causes cavitation, solution foaming and results in protein particle formation.

This body of knowledge has grown considerably over the last decade, and much work was stimulated by participants from the first Workshop on Protein Aggregates and Immunogenicity Conference in 2006, who identified an analytical gap in assessment of particles in therapeutic protein products. Most products were not being analysed for particles smaller than 10 µm. Since then, the increasing focus on proper and rigorous analysis of subvisible particles has attracted attention of laboratory instrumentation manufacturers who are aiming to provide bioopharmaceutical scientists with the high-tech solutions needed to gain insights into causes and control of particles and resulting increases in product quality.

Detection, identification and characterisation When considering the level of technology available for detecting, identifying and characterising these particles back in 2006, biopharmaceutical scientists and the laboratory professionals who serve the industry in the US were mostly following USP<788> guidelines for particulate matter in parenteral products. This guideline required, and continues to require, drug manufacturers to count and size subvisible particles >10 µm. This size range was based on concerns about occluding capillaries in patients with particulate matter. USP<788> testing typically depends on light obscuration, with microscopy providing another option. For protein particles, light obscuration is notoriously insensitive to even particles >10 µm; often resulting in undercounting and/or mis-sizing particles.

So, in 2006, not only were most product quality assessments ignoring particles in the important sizes less than 10 µm, but with light obscuration, protein particles were also often greatly under counted. Although passing USP<788> is necessary for all parenteral products, for therapeutic protein products this level of quality assurance is not sufficient.

However, even today, many companies try to use only USP<788> and light obscuration for assessment of particles in therapeutic protein and peptide products. Such limited particle characterisation and the resulting limited understanding of factors causing particles, often leads to unanticipated problems. Such problems include quality failure due to formation of visible particles and adverse events in patients such as immunogenicity and infusion reactions.

In one recent tragic case with Peginesatide (Omontys®; Affymax, Inc., Cupertino, CA), seven fatalities and 49 cases of anaphylaxis were reported soon after commercialisation in 2012. The product had passed USP<788> testing using light obscuration and all other lot release assays. The drug was voluntarily withdrawn from the market. To investigate the potential cause(s) of the product safety problems, both the FDA and the company repeated the USP<788> assays only to find the product met approved release specifications. No new impurities or contaminants were found. Then further testing was conducted of some vials from commercial lots and from clinical lots for nanoparticles and microparticles. Nanoparticles were characterised with the NanoSight instrument and microparticles were studied with flow imaging microscopy using the FlowCam instrument. Much higher levels of both nano- and microparticles were observed in the problematic commercial product, than in the clinical trials material, which was safe and effective. Such results document the importance of choosing the appropriate methods for characterising and counting both nano- and microparticles to assure the safety of therapeutic protein products and to assure that product quality is reproducible from lot-to-lot³.

The microparticles were properly quantified because the FlowCam operates differently than traditional light obscuration instruments. It automatically takes a high-resolution, digital image of each particle detected, whether opaque, translucent or transparent, and simultaneously determines sizes and concentrations of particles. Measurement data using more than 40 different parameters from size and color to morphological characteristics such as circularity, elongation and fiber curl, is based on the actual image of each particle. These images yield highly accurate data, and allow the company's proprietary pattern recognition software to aid in identifying the particle type by differentiating, for example, circular silicone oil droplets from glass shards and irregularly-shaped protein particles. Further, the latest model automatically detects, images and helps identify nanoparticles and microparticles in a fluid stream from 300 nm to 10 µm at the same time. This offers drug manufacturers an improved method for testing product quality and reproducibility of the manufacturing process than can be obtained with traditional USP<788> testing with light obscuration.

Time for change Now that the technology to quickly quantify and characterise the different types of submicron and micron-sized particles that may be present in therapeutic protein formulations exists, do drug manufacturers bear additional responsibility to find and characterise these particles ­– even though the USP requirements remain unchanged?

In some published and in numerous unpublished studies, relying exclusively on USP<788> guidelines and light obscuration for characterisation of particles in therapeutic protein products has led to many problems, ranging from severe adverse effects in patients, to failed clinical trials to manufacturing holds because of unexpected appearances of visible particles. In each of these cases, critical insights into the causes of the associated product quality problems have been obtained by using the modern methods for characterisation of nano- and microparticles. It is important that these methods be adopted early in product development and used throughout a product’s lifetime. And using orthogonal methods, which detect particles by different technologies, helps to ensure that the analytical methods are robust and reliable.

Companies that are taking these approaches are minimising the risk of product safety problems and quality failures, and ensuring the higher quality products are consistently delivered to patients.

References:

1. Neha N. Pardeshi, Wei Qi, Kevin Dahl, Liron Caplan, John F. Carpenter. Microparticles and Nanoparticles Delivered in Intravenous Saline and in an Intravenous Solution of a Therapeutic Antibody Product, Journal of Pharmaceutical Sciences, 106 No. 2 (February 2017): 511-520.
2. Anil K. Tyagi, Theodore W. Randolph, Aichun Dong, Kevin M. Maloney, Carl Hitscherich Jr., John F. Carpenter. IgG     particle formation during filling pump operation: A case study of heterogeneous nucleation on stainless steel nanoparticles, Journal of Pharmaceutical Sciences, 98 No. 1 (January 2009): 94-104.

3. Joseph Kotarek, Christine Stuart, Silvia H. De Paoli, Jan Simak, Tsai-Lien Lin,Yamei Gao, Mikhail Ovanesov, Yideng Liang, Dorothy Scott, Janice Brown,Yun Bai, Dean D. Metcalfe, Ewa Marszal, Jack A. Ragheb. Subvisible Particle Content, Formulation, and Dose of an Erythropoietin Peptide Mimetic Product Are Associated With Severe Adverse Postmarketing Events, Journal of Pharmaceutical Sciences, 105 No. 3 (March 2016) 1023-1027.

 

Author:  Dr John F. Carpenter, is Professor of Pharmaceutical Sciences and Co-Director of the Center for Pharmaceutical Biotechnology at University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences.