E&L Risk Assessment for Biologic Drug Products

Published on: 
Pharmaceutical Technology, Pharmaceutical Technology-08-02-2018, Volume 42, Issue 8
Pages: 48, 50, 52

Materials in contact with a drug must be fully characterized to ensure they do not negatively affect the safety and efficacy of the product.

The packaging or container closure system that is meant to protect a pharmaceutical product can be a source of contamination. Comprehensive extractables and leachables (E&L) studies are, therefore, required to identify and quantify harmful impurities that could affect the quality and safety of drug products.

Pharmaceutical Technology spoke with Lester Taylor, Pharma marketing manager, Agilent Technologies; Andrew Blakinger, manager, Extractables and Leachables Testing, Eurofins Lancaster Laboratories; and Fran DeGrazio, vice-president, Global Scientific Affairs and Technical Services, West Pharmaceutical Services, about the ins and outs of extractables and leachables assessments in biologic drug products.  

PharmTech: What are the E&L challenges for biologics compared to small-molecule drugs?

Taylor (Agilent): Compared to small-molecule drugs, biologics face additional challenges. For example, the efficacy of a biologic drug may potentially be reduced through undesirable interactions of leachables with drug molecules through post-translational modification (PTM) biochemical reactions (e.g., oxidation, aggregation, clipped variants, unfolding, adducts formation, and glycosylation). Alternatively, a leachable arising from single-use systems (SUS) or components used for bioprocessing may adversely affect the manufacturing process through cellular toxicity and Chinese hamster ovary (CHO) cell death thereby reducing the productivity of the bioprocess. There are several examples where leachables have been associated with these undesirable effects on biologic manufacturing and drug efficacy, leading to major manufacturing losses and even worse, dangerous side-effects and loss of drug efficacy. 

Blakinger (Eurofins): The evaluation of biologics for leachables presents many unique challenges. The protein itself can interfere with testing, so removal prior to analysis may be warranted. But if care is not taken, this process can unintentionally remove potential leachables, resulting in false negatives, or it may lead to contamination of the sample that may result in the generation of false positives. 

Other ingredients in large-molecule formulations, such as polysorbate 80 and other surfactants/stabilizers, can also cause issues. These compounds often interfere with chromatographic analyses in the form of multiple large peaks that display numerous ions by mass spectrometry throughout the retention time window. These large surfactant peaks can easily mask leachables. Furthermore, proteins, surfactants/stabilizers, and other ingredients in large-molecule formulations are difficult to clean from mass spectrometers and, therefore, may carry over from one analytical run to the next if not dealt with properly. 

DeGrazio (West): The likelihood of leachables in any drug product will depend on the packaging materials, type of formulation ingredients, and conditions of use. The occurrence and impact of leachables in biologic products can present greater challenges compared to that of small synthetic molecules due to several factors. Biologics are living molecules that can be difficult to solubilize and stabilize, and quality attributes are not easily characterized compared to small molecules. The formulation ingredients for biologics often contain co-solvents or surfactants and will have more propensity to extract chemicals from packaging materials compared to typical small-molecule formulations. 

Biologic products are complex and very sensitive to their environments. Extractables or potential leachables that may migrate into a drug product have the potential to interact, and therefore, affect the product quality, safety, or stability. In general, biologic products are formulated to solubilize, stabilize, and optimize pharmacokinetic properties consistent with the route of administration. Anything that migrates from the packaging that could interfere with this optimized environment is of concern. This includes interactions with active or excipients in a drug product formulation that lead to said quality, safety, or stability issues.

Additionally, large molecules have greater surface areas with sites that have a propensity for interactivity based on polarity and charge. This can lead to conformational modifications and other interactions that may impact product quality.


Primary packaging and container closure systems

PharmTech: What are the key considerations when selecting primary packaging material for biologic drug products?

DeGrazio (West): With every drug product and especially biologics, the most inert primary package possible must be chosen to minimize the potential for interactions to occur. Potential leachables are not the only interaction of which to be wary. Because of their reactive nature, biologic drug products can adhere to surfaces or absorb into materials. An understanding of possible interfacial interactions must be a consideration. In addition, there are other packaging considerations that must be addressed, such as container closure integrity, particle generation, and other performance concerns.

Blakinger (Eurofins): For any drug product, it is crucial to ensure the packaging does not adulterate the drug product. Any compounds that leach from the packaging could affect the product in a variety of ways, including impacting patient safety if compounds are toxic or interfering with other analytical assays during release testing. 

There are a number of other potential E&L risks that are unique to large molecules. Leachables may cause conformational changes in the protein or may cause the protein to aggregate. Large-molecule drug products may also chelate inorganic leachables. These types of interactions can increase the toxicity of the drug product, reduce the product’s efficacy, or affect the product’s stability. It is, therefore, important to fully evaluate the E&L risks to avoid costly delays in getting a product to market. 

PharmTech: What components in a container closure system can pose E&L risks to a biologic drug product?

Taylor (Agilent): Typically, the container and closure components that come into direct contact with the drug product usually have the highest impact in terms of leachables observed. However, there have been many examples of leachables arising from package labels such as the inks or adhesives, as well as from secondary packaging components. These risks should, therefore, be assessed during bioprocess development.

Blakinger (Eurofins): Nearly any component in a container closure system may pose E&L risk to a biologic. Because many biologics are packaged in prefilled syringes, some of the most common components of concern are rubber stoppers. Rubber stoppers are notorious for containing nitrosamines and polynuclear aromatic hydrocarbons (PAHs), both of which are carcinogenic. Glass prefilled syringes are another common example of a component type posing a special risk to biologics. During manufacturing, tungsten pins are used to hold open the fluid path in the syringe barrel. Because manufacturing occurs at extremely high temperatures, the formation of tungsten oxides is possible. The residual tungsten oxide on the glass syringe can then leach into the final biologic drug product and cause protein aggregation or degradation. 


DeGrazio (West): The most common primary packaging system for a biologic drug product is a vial system. This system is composed typically of a glass vial with an elastomeric rubber stopper and an aluminum seal with a plastic flip-off button. The other common primary package is a prefilled syringe system, which is typically a glass syringe with an elastomeric plunger and a tip cap or needle shield. Each of these components has the potential to leach substances into a drug product with contact over time. Of course, the extractables of most significant concern from glass materials are metal ions. It is well known that some biologics drugs are sensitive to various metal ions. Although these reactions are drug-product specific, these reactions are a consideration when evaluating packaging components.

Other types of extractables are expected from elastomeric components. Elastomeric components are composed of much more than just the base polymer. Elastomer formulations typically have six to 12 added ingredients that are mixed with the base polymer under heat and pressure. This process causes chemical crosslinking to occur, which result in the formation of reaction products. These reaction products, along with residual compounds of the original raw materials, may interact with the active drug product or environment. Many of these compounds are organic; some may be inorganic, and, therefore, provide an additional source of metal ions.

In the case of a prefilled syringe system, there is the potential for even more extractables. A glass syringe may be formed with the use of a tungsten pin. This can result in tungsten residuals that are known to interact with proteins. Another issue is that syringes typically use silicone oil as a lubricant for easier plunger movement. Silicone oil can migrate into the drug product and silicone oil droplets can act as a nucleus for particle formation/growth and protein aggregation.

Newer packaging components are now being introduced to the industry; for example, engineered polymers are replacing glass. These polymers, such as cyclic polyolefins, are much lower in extractables and have lower surface tension characteristics that make them suitable for biologic drug products.

PharmTech: Why is it important to fully characterize contact materials and understand the material of construction for the container closure system and their associated E&L?

Blakinger (Eurofins): Fully characterizing contact materials is crucial to ensure the materials chosen do not negatively affect the safety or efficacy of the drug as a result of leachables. Ideally, multiple options for container closure systems should be evaluated during the initial extractables screening. Then the packaging with the lowest risk can be selected. Establishing an extractable compound profile helps to ensure that the observed compounds are not overlooked during subsequent leachables evaluations. The constituents of large-molecule drug products often interfere with the analytical tests used to evaluate E&Ls. By establishing a material’s extractable profile, leachable analysis by mass spectrometry, using extracted ion analysis, can specifically target those compounds to evaluate their presence in the drug product. This technique effectively eliminates any matrix interferences and ensures leachables are not overlooked.


Risk assessments

PharmTech: What assessments should be performed to evaluate the potential risks of E&Ls from primary packaging that meets the biologic drug product?

DeGrazio (West): It is important to take a risk-based approach to choosing and evaluating the packaging components. It should start with supplier information on the components or system, addressing questions such as:

  • What are the basic material characteristics of the components?

  • Are there special needs associated with the biologic drug product application, such as the environmental conditions of storage?

Once this information is gathered, basic evaluation by standard compendial methods is needed for compliance and allows one to begin to ‘qualify’ a component for use. But this is only the first step in proving suitability. Once compendia requirements are passed, material characterization is essential to better understand what may be extracted from the material (at levels critical to the drug product).

The following highlights the best practice recommendation for addressing E&L for a primary package:

  • Material characterization: Each individual component should be assessed to assure it has broad applicability for the application.

  • Controlled extractables study: This study is a comprehensive program to understand what could be extracted from the components under a broader series of solvents, if the material characterization information is found not to be sufficient. It is crucial to perform a risk assessment to decide if this step is needed, and to determine the appropriate next step in the process based on the application. The solvents used should be aqueous-based, with considerations for organic solvents (if needed), pH, extraction conditions (such as time), extraction methods, material-to-solvent extraction ratios, etc.

  • Simulation study: Depending on the drug product application, it may be appropriate to complete a simulation study, instead of going directly into a leachables study. This study is highly probable when it is especially challenging to reach the analytical evaluation threshold (AET). This may occur in a circumstance such as when evaluating a large-volume parenteral (LVP) application where there is a significant volume of drug solution. If many extractables are found from the controlled extractables study, the simulation study is a way to help identify the probable leachables to target in a formal leachables study.

  • Data assessment: To determine the targets for a leachables study, it is important to evaluate the risk in the specific drug application.

  • Leachables study: Method development and validation for specific leachables in the drug product should occur. Leachables testing should be conducted over drug product shelf life, at both room temperature, and accelerated conditions. The leachables should be identified based on the safety concern threshold (SCT). The SCT is the threshold dose below which a leachable would present negligible safety concerns for carcinogenic and noncarcinogenic effects. The recommended SCT for parenteral drug products, per the Product Quality Research Institute (PQRI) Extractables & Leachables Working Group for parenteral and ophthalmic drug product (PODP), is 1.5 ug/day (as described in an April 2018 workshop). 

  • Special considerations for biologics include: biologic activity, efficacy, degradation, oxidation, chemical modification, immune adjuvant activity. 

Taylor (Agilent): Typically, the first step is to perform an extractables profiling study on the packaging component of interest to identify the potential list of leachables in the drug formulation. The profiling study results may be used to perform a risk assessment with two goals: 

  • To identify potential ‘bad actors’ from the list of extractables through predicting toxicity or performing toxicology experiments 

  • To select components that have more desirable extractables profiles for the final process and eliminate components found to likely contribute to an undesirable leachable.

PharmTech: How do you identify and quantify potential E&L from container closure systems?

Blakinger (Eurofins): The first step is to expose the components of the container closure system to several model extraction solvents at exaggerated conditions of time and/or temperature. The resulting solutions are then screened by headspace and direct injection gas chromatography–mass spectrometry (GC/MS) for volatile and semi-volatile organic compounds, liquid chromatography–mass spectrometry time of flight (LC/MS–TOF) for non-volatile organic compounds, and inductively coupled plasma-mass spectrometry (ICP/MS) for elemental impurities. Additional testing methods may be used if appropriate, such as those specific for halide ions, nitrosamines, or PAHs. At Eurofins, we use the Wiley/National Institute of Standards and Technology (NIST) databases to identify compounds detected by GC/MS. For those compounds detected by LC/MS, we have a propriety database, the Eurofins Extractables Index, containing more than 1500 non-volatile organic compounds. If a compound cannot be identified via the database, additional testing may be necessary. Not only does this additional testing require advanced instrumentation (e.g., quadrupole time of flight [Q-ToF]), but it also requires the expertise of experienced and highly educated analysts.

Taylor (Agilent): Establishing a holistic extractables profile for an article of interest is a complex and intensive process involving the use of a variety of analytical technologies. Gravimetric studies and total organic and inorganic carbon analysis are often performed to gain an understanding of the total extractable content. Fingerprinting of extracts using spectroscopic methods such as ultraviolet–visible spectroscopy (UV–VIS) and Fourier transform infrared spectroscopy (FTIR) resulting in generic information about constituent chemical classes is also common. 

These methodologies are typically followed by more specific qualitative studies to identify volatile, semi-volatile, and non-volatile extractables using GC/MS and LC/MS techniques respectively (including high resolution accurate mass [HRAM] determination). Compounds are usually identified above the (AET) that has been determined for the material or article of interest. The AET for an article of interest depends on the target dose and number of doses expected to be stored in the container closure system or component. Analytically, the AET is used to estimate a detector response threshold using a set of reference standards carefully selected to represent the chemicals expected to be extracted. Once a list of extractable peaks above AET is identified, relative quantitation is also performed to better inform risk assessment.

In parallel, it is also important to identify any elemental impurities that result from the extractions. This [assessment] is usually performed through either inductively coupled plasma optical emission spectrometry (ICP–OES) and inductively coupled plasma mass spectrometry (ICP–MS) methods depending on required specificity and sensitivity.

PharmTech: Which analytical techniques are robust enough to identify potential E&Ls?

DeGrazio (West): There is no one method that will identify all potential E&Ls. Multiple analytical techniques are needed for comprehensive assessment of extractables and leachables. For inorganic species, ICP/MS or OES are typically employed. GC/MS and LC/MS are the most common techniques for detection and identification of organic compounds. There are various LC/MS configurations for robust non-volatile organic analysis. Various additional features/techniques can improve sensitivity. One such example is ion mobility and Q-ToF to enable more precise analyses and identification of unknowns by combining ion mobility and mass-to-charge ratio.

Article Details

BioPharm International
Vol. 31, No. 8
August 2018
Page: 48, 50, 52


When referring to this article, please cite it as A. Siew, “E&L Risk Assessment for Biologic Drug Products," Pharmaceutical Technology (8) 2018.