By James Netterwald, Ph. D.
Due to their therapeutic benefits, biopharmaceuticals already represent a significant portion of drugs in the global pharmaceutical pipeline and are fast becoming even more predominant. Many biopharmaceuticals are antibodies and thus, are composed of high concentrations of protein molecules.
The high propensity of protein molecules in biopharmaceuticals to aggregate into subvisible particles is a concern for both the FDA and the biopharmaceutical companies that produce these monoclonal antibody-based drugs, thus prompting industry and regulators alike to take notice of this growing problem.
The adverse health effects associated with particle contamination in parenteral (or injectable) drugs have been known for decades. Since the 1980s, automatic particle analyzers based on the principle of light obscuration have been used to detect these particles. The particles can originate from a variety of sources, including employee gowns, rubber closures, or airborne dust, and are collectively termed “foreign contaminants.” The presence of these particles will result in entire drug lots being discarded during the quality assurance process.
In addition to foreign particles, the FDA also has known about the presence of protein aggregates in biopharmaceuticals for several years. “The FDA, and other subject matter experts have suspected that these protein particles may affect the safety and efficacy of the drug itself. This interest from the FDA and the industry’s inherent commitment to product quality have created an interesting opportunity for instrument manufacturers,” says Peter Moore, president of Brightwell Technologies.
The main safety concern with protein aggregates is immunogenicity. In other words, the presence of protein aggregates in biopharmaceuticals can elicit an immune response in the patient, which could eventually render the drug ineffective. “Those of us working in the field of protein aggregation and concerned with immunogenicity therapeutic proteins realized that particles smaller than 10 µ could be important for both quality attributes as well as potentially immunogenic,” says John Carpenter, Ph.D., professor at University of Colorado, Anschutz Medical Campus in Aurora, CO. With this discovery, it is fast becoming more routine practice to quantify particles as small as one to two microns. The mass of protein in these small particles is usually too small to allow detection of loss of monomer by methods such as size-exclusion chromatography.
“So, partly from product quality standpoints and partly from concerns about immunogenicity, people have started to look at these particles. What we’ve started to learn is that by counting the smaller particles, we have a very sensitive method to follow protein aggregation,” says Carpenter. The field is now spending more time and energy trying to understand how and where these particles are generated, as well as what roles they might play in product quality and immunogenicity. The industry has also been developing methods and instruments to count these particles directly.
Detecting Protein Particles
With respect to biologic drugs, the limitation of light obscuration analyzers is that, although they reliably detect foreign contaminants, they do not detect protein aggregates (also known as intrinsic particles) in biopharmaceuticals. The reasons behind this deficiency relate primarily to the semitransparent and highly heterogeneous nature of the particles, the underlying optical technology, and the method of calibration. However, for the FDA and biopharmaceutical manufacturers, the salient fact is that these protein aggregates cannot be reliably measured using traditional techniques. Furthermore, due to the immunogenic significance of aggregates and particulates, the FDA has stated that they pose a clear risk to the safety and efficacy of protein therapeutic products and are therefore considered a critical quality attribute. It has led to a comprehensive investigation of alternate measurement technologies.
“These so-called intrinsic particles, which often include protein aggregates, silicone oil droplets, and air bubbles, are very difficult for traditional methods to accurately quantify. That difficulty has created strong interest in new technologies which can be applied to this problem. Flow microscopes have been widely studied and have emerged as some of the leading technologies to help measure these subvisible particles,” says Moore.
Flow microscopes use digital imaging technology to measure these challenging particles. Using optical components similar to a standard digital microscope, a flow microscope includes special fluidic components and software to measure particles suspended in fluids. In addition to being able to detect semitransparent particles like protein aggregates, the images from a flow microscope allow formulation scientists to analyze the shape of the particles, which has proven to be an added bonus for the pharmaceutical industry.
“The problem from the patients’ perspective is that they take these therapies for conditions such as Crohn’s disease or multiple sclerosis, and the products control their disease. And, then after a while for a given patient, the product might quit working. Those patients become nonresponders because they have become immune to the product. In other words, the product is acting like a vaccine,” says Carpenter. So how does this happen even after these products have been deemed by the FDA as safe and effective?
“We don’t know why these drugs quit working and what causes the immunogenicity,” says Carpenter. “The problem is that you can’t ethically run a human trial to test if protein particles are the problem. The industry acknowledges that protein aggregates and particles are present in drug products, and companies share the concerns about nonresponders and immunogenicity. But it is difficult to establish, clinically, a definitive link between protein aggregates/particles and nonresponsiveness in patients.”
Pharmaceutical companies are concerned about the issue because the loss of efficacy means that patients are no longer benefitting from the therapeutic protein product. Also, if enough patients become nonresponders to a company’s drug, then that company will start to lose market share. “I think it is very important for pharmaceutical business managers to realize that when patients become nonresponders, they’re basically taking a product that has no value and costs thousands of dollars,” says Carpenter. “I think eventually the payers (health insurance companies) could require physicians to check patients for both drug level as well as for the presence of antibodies against the drug. At the point the drug is no longer detectable because of antibodies, the payer would not pay for the drug anymore.”
Impact Of Regulatory Requirements
Carpenter says there is no way to eliminate the particles completely, but industry and academic researchers are working to understand the causes for particle formation during processing and in the final drug product. This insight will be used in attempts to control protein aggregation so that particles and other aggregates are kept at a low level. “The most important thing is to know that these aggregates are present and to be able to assay them properly,” says Carpenter. Companies must ensure that the levels of protein aggregates and particles in commercial batches do not exceed those present in batches administered to patients during clinical trials. Carpenter says that although pharmaceutical companies can set some limits for each type of particle, it is extremely difficult to prove whether a given type of particle or other degradation product is responsible for safety or efficacy issues in patients. The FDA and pharmaceutical companies are working toward developing guidelines to ensure the level of protein aggregates and particles in commercial batches do not exceed the levels present in the drug product during clinical trials, but the industry is not there yet.
Right now the FDA wants to know how well companies are testing for particles in their commercial batches. “However, there are currently no FDA guidelines for specific protein degradation products. And that is done on purpose,” says Carpenter. “It is not possible to create specific guidelines that are applicable to all product drugs because each protein is unique. The FDA cannot set limits on degradation products because they just really don’t know what limits to set; it really depends on the impact a given degradation product has on safety and efficacy, on the kind of product, and how often it is administered.”