Metal Leachables in Therapeutic Biologic Products: Origin, Impact and Detection

• Pfizer

Introduction

Pharmaceutical companies invest significant resources to identify, quantify, and minimize impurities in their drug products. An area of increasing concern and scrutiny from regulatory authorities is the potential adulteration by compounds that may migrate into the drug product through contact with components and materials used in manufacturing, storage, and delivery of the therapeutic. These compounds have been typically referred to as extractables and leachables.  An extractable and a leachable are defined as follows: “the term extractable is specific to a particular container/ closure material. A compound is said to be an extractable if it is identified in solvents that were exposed to virgin container material under standard conditions, as specified in the USP. A compound is said to be a leachable if it is identified in a drug product following storage of that product, but was not identifiable in the product initially (assuming the compound is not a degradation product). Therefore, an extractable is a theoretical impurity of a drug product that utilizes a container/closure material known to be associated with that extractable compound, while a leachable is known impurity of a given product [1]”. Leachables are typically a subset of extractables.

Contamination by leachables raises concerns about the safety and efficacy of the product. They have the potential to cause acute toxicity or long-term health issues through chronic exposure. An example of such a compound is Bisphenol A that has recently come up for extra scrutiny [2]. Leachables can also potentially negatively impact product quality by interacting with the active substance or excipients leading to altered physico-chemical properties as well as reduced stability and shelf-life.

In the US, the requirement for the proper evaluation of extractables and leachables (E&Ls) is governed by 21 CFR parts 210 and 211. Current Good Manufacturing Practice in Manufacturing, Processing, Packing or Holding of Drugs: General and Current Good Manufacturing Practice for Finished Pharmaceuticals, Subpart D-211.65 which states: “Equipment shall be constructed so that surfaces that contact components, in-process materials, drug products shall not be reactive, additive, or adsorptive so as to alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements.”

Specific guidance has been issued by the regulatory authorities for monitoring and control of leachables as part of requirements for the primary packaging of drug products [1, 3]. The degree of concern is highest for orally inhaled and nasal drug products as well as injection (solution and powder) products. The likelihood of interaction between packaging and dosage form is considered high for solutions or suspensions and medium for powders. The majority of biological proteins are formulated as liquid or freeze-dried products and are delivered via injection, and therefore fall into this category of highest concern.

Compared to small molecules, proteins in nature exhibit significantly lower stability due to the strong dependence of their physico-chemical properties on their structure and conformation. This and their generally larger-size offer multiple sites for potential interaction with a leachable, increasing the risk for degradation and loss of activity [4]. As a consequence, the impact of extractables / leachables on biotherapeutic products may be more significant and also more complicated to assess than those for small molecules.

The three main categories of contact materials in the biopharmaceutical drug product manufacturing process and storage include: plastics/elastomers, glass and stainless steel. Plastics/ elastomers are by far the biggest source of extractables/leachables (E/ Ls) and present significant analytical challenges for their detection, identification, and quantitation. E/Ls from plastics/elastomers arise from monomers/oligomers, plasticizers, crosslinking agents, curing agents, antioxidants, other additives such as adhesives, colors, fillers, and inks etc., and the degradation products of these compounds [4- 7]. A number of publications are available that provided strategy and guidance for selecting, evaluating, qualifying appropriate container-closures as well as strategies for safety evaluation [8-11]. E/Ls from other contact surfaces have also been studied [12-14]. On the other hand, metals are the primary leachables from glass and stainless steel. Metal leachables are generally easier to analyze (compared to those from e.g. plastics) but can also have profound impact on biological protein products, and are the subject of this short review. This paper will address the origin of metal leachables, their impact on protein product quality / safety and the analytical tools to identify and quantitatively measure them.

The Origin of Metal Leachables

Stainless steel is a typical contact material used during the manufacturing, shipping, and storage process for biologic products. Stainless steel commonly used in biopharmaceutical applications is of the grade 316L and is an alloy containing mainly iron, nickel, chromium with minor amounts of manganese and vanadium. Stainless steel is a major source for metal leachables, especially if the surface of the equipment or tank is not properly treated. The main leachable components are iron, chromium and nickel. Several fold higher levels of metals such as iron and nickel have been shown to leach into a liquid formulation after storage at room temperature in unpassivated compared to passivated stainless steel vessels [12].

Even though stainless steel is the major source of metal leachables during processing, metals can also leach from various contact materials such as glass vials, rubber stoppers and other product-contacting equipment in the drug product manufacturing and storage process. Less frequently, it has been shown that some plastic packages can also be a source for metal leachables [12].

Waterman et al [15] and Fliszar et al[16] have demonstrated that greater amounts of manganese and iron ions could leach from amber glass compared to clear glass. Manganese and iron oxide are present in Type I amber vials to act as coloring agents. Typical levels of iron oxide in Type I molded amber vials range from 1.0-1.24% in contrast to less than 0.05% in Type I molded clear vials. Levels of manganese oxide in amber vials range from 5-7% [17]. Zinc and barium oxide oxides are also present in Type I glass. Bohrer et al [18] indicated that different sterilization treatments impacted the amount of metal leachable levels from glass vials. Steam autoclaving, the typical sterilization technology used in biopharmaceutical manufacturing, tends to substantially increase metal leachables from glass vials. Gamma irradiation of glass containers tends to form larger amounts of radical species and organohydroperoxides, but produces less metal leachables compared to steam sterilization.

The drug product dosage form and its composition also play an important role in metal leachables. When the drug product in liquid formulation contains chelating agents or excipients that might help plasticize or wet the plastic, trace amount of metal ions such as aluminum, calcium, iron, manganese and zinc can leach from plastic packages [16]. Kocijan et al [19] found that proteins and complexing agents such as EDTA facilitate the migration of metal ions into solution from metal contact surfaces. Proteins varied in their ability to leach metal ions. Significant dissolution of the ions was seen in solutions containing EDTA, simply due to its high metal complexing ability. Furthermore, the ability of EDTA to leach metal ions far exceeded that of protein. Buffer agents such as phosphate or lactate, and antioxidants can also accelerate metal leaching from glass or stainless steel [12]. Trace amounts of the residual metals in formulation buffers or other excipients is another potential source of metal ions.

The Impact of Metal Leachables on Biologic Products

Proteins can have significant interactions with metal ions. In vivo, about one third of the known proteins require metal ions to perform their functions [20, 21]. In general, metal ions either binds to catalytic sites to regulate the protein functions or bind to structural sites to stabilize protein structure or induce conformation changes [22]. Alkali and alkaline earth metals bind proteins predominantly through electrostatic interactions while the transition metals, such as Fe (II/III), Co (Ii), Cu(II), Al(III), and Mn(II), covalently bind to proteins [22]. The most common ligands are sulfur, nitrogen, and oxygen atoms, including backbone carbonyls. At the amino acid level, the most common metal-binding residues are cysteine, histidine, aspartic acid and glutamic acid. The coordination numbers vary greatly among different metals ranging from one to eight. The coordination varies with pH and the ionization state of the amino acid residue. Other residues that may contribute to the coordination include tryptophan, tyrosine, phenylalanine, arginine, methionine and glycine. Proteins can also function as chelates if there exist a number of carboxyl groups in close proximity so as to form complexes analogous to those by EDTA. These complexes will also be dependent on pH.

Although many protein-metal complexes have an important role in biological systems, the inadvertent contamination of biotherapeutics products with metal ions can have a profound impact on the stability of these products. Trace levels of metal ions can cause degradation via different mechanisms, such as protein oxidation [23-26], fragmentation, aggregation [23, 26, 27], or the formation of insoluble particles. Metal-catalyzed oxidation of proteins occurs via site specific interactions [28, 29]. The oxidized amino acid residues are either directly involved in metal-binding or located in close vicinity to the metal binding site. The side chains of histidine, methionine, cysteine, proline, arginine, lysine, tryptophan and tyrosine, are sensitive to metal-catalyzed oxidation due to their high electron densities. Among them, methionine and histidine are two of most susceptible residues. Methionine oxidizes to form methionine sulfoxide or methionine sulfone while histidine is predominantly converted to 2-oxo-histidine.

Site specific metal-protein binding can also induce secondary and tertiary structure changes resulting in the formation of protein aggregates [30, 31]. In vivo, heavy metals have been implicated in the aggregation of proteins responsible for several neurodegenerative diseases such as Alzheimer and Parkinson diseases [27].

 Trace amount of transition metal ions were also found to induce protein fragmentation [24, 26]. Aromatic side chain, tryptophan, tyrosine and histidine are subject to cleavage. The cleavage occurs at the metal-binding sites via the Fenton reaction. Proteins that are exposed to oxygen radicals result in modification of amino acid residues leading to extensive fragmentations [32]. Metal-induced fragmentation is more specific and less extensive than that induced by oxygen radicals due to the high diffusivity and energetics of the latter.

Markovic has provided several examples where the quality of protein drug products was negatively impacted by metal leachables [14]. Metal cations migrated from rubber stoppers into a therapeutic protein liquid formulation inducing protein N-terminal degradation. Barium leached from glass vials reacted with sulphate in the formulation to form visible particles. Salts of tungsten oxide migrated from prefilled syringes into the drug product and triggered protein oxidation followed by aggregation [33]. In summary, the impact of metal ions on biologic products exhibits a complex dependence on the physical-chemical structure of the protein of interest. Due to the complexities of the metal interactions with protein, the impact of metal ions on protein should be carefully evaluated during development on a case by case basis.

Spiking studies with metal ions, such as FeCl3 can be used to investigate the sensitivity of a protein to metal-induced degradation. If the product is found to be sensitive to metal leachables, there are several strategies that can be utilized to mitigate their impact. The simplest is by the addition of various chelating agents to the formulations (e.g., cell-impermeable chelators like CaNa2EDTA, dipicolinic acid (DPA), or diethylenetriaminepentaacetic acid (DTPA) [26, 34] and cell-permeable chelators such as pyrophophates [35]. Although, the chelating agent can enhance the extraction of metal ions, they can also sequester these ions due to their high comlplexing ability. However, care must be taken since in certain cases, formation of EDTA-Fe3+ complex can eliminate site specific oxidation, but still allow non-site specific degradation to occur through the generation of reactive oxygen species [36]. Also, if metal induced oxidation is initiated and propagated via a chain reaction, addition of chain terminators may be an alternative way to decrease the degradation. Buffers such as histidine and citrate also have certain metal chelating capacity.

Other strategies would include reduction of metal contact with welded and/or non-passivated stainless steel during drug substance/ drug product manufacturing, processing and/or storage. Also useful is limiting exposure to stainless steel surfaces during low-pH processing steps and in the presence of chloride ions [37, 38]. High purity excipients and buffer salts can also be used for metal-sensitive biologic products. Higher grade alloys can also be used in manufacture or storage but such systems are expensive to fabricate and maintain [39, 40].

Analytical Tools to Quantitate Metal Leachables

A reliable and sensitive analytical method is very critical to detect and determine the concentration of the metal leachables in the biologic products. This can be difficult due to complex matrices and the trace amount of the metal ions in the biologic products. Two high sensitivity methodologies for metal analysis are Inductively Coupled Plasma - Mass Spectrometer (ICP-MS) and Atomic Absorption Spectrometry (AAS). Atomic spectroscopy can be used to detect more than 60 elements in the periodic table. AAS was adopted to analyze the transition metal concentration of zinc, cobalt, nickel, copper and iron in Ceruloplasmin solution and its dialysis buffer solution [41]. The Atomic spectroscopy technique is highly selective and sensitive for liquid samples and can be used for qualitative and quantitative determination of inorganic leachables. However, transitions between energy levels produce multiple spectral lines which can create spectral interferences in complex samples. The detection and measurement of analytes in solution is dependent on the sample matrix and analyte solubility. ICP coupled with MS has been widely used in lieu of the traditional atomic spectroscopic techniques due to its superior sensitivity and selectivity. ICP-MS can measure most of the elements in the periodic table. The typical metal ions of interest in biologic products (e.g., Fe, Co, Ni, Cu, Zn, and Mn) can be analyzed by ICP-MS with detection limit at or below the part per trillion (ppt) range. Due to extreme high temperature of the plasma ion source (which can completely break apart the molecules in a sample) ICPMS detects only elemental ions. This is very comparable to Atomic Absorption Spectroscopy. However, ICP-MS offers detection limits that are equal to and sometimes better than Graphite Absorption Furnace Atomic Absorption and with higher productivity. Also, ICPMS can easily handle both simple and complex samples.

Conclusions

Leachables pose a toxicological and stability concern for biological drug products. Metal ion leachables from manufacture and storage can interact with proteins and lead to oxidation, fragmentation or aggregation. Strategies are proposed for addressing these if a product is found to be sensitive to such contamination. AAS and ICP-MS are the analytical tools available to detect and quantify metal ions in products. Impact of metal ions must be evaluated as part of formulation and process development of biologics.

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Shuxia Zhou, Shuxia Zhou, is a Scientist, Biotherapeutic Pharmaceutical Science, Global Biologics at the Pfizer Corporation (formerly Pharmacia). She earned her B.S. from Fudan University (China) and M.S. from University of Mississippi (USA) in Analytical Chemistry. She has been involved in various analytical chromatographic method development. Her current interest is in formulation, process and product development for various liquid/lyophilized biologics products. She also serves as representative of PhRD at Pfizer E&L network and is actively involved in the investigation of metal extractables and leachables, such as formulation factors affecting metal leachables, metal impact on biologics product and strategies to reduce the adverse effect.

Lavinia Lewis, Ph.D. is a Senior Principal Scientist and Group Leader at the Pfizer Corporation (formerly Pharmacia). She earned her Bachelors in Pharmaceutical Sciences in 1993 from the University and Department of Chemical Technology, India; her Masters in Pharmaceutics (1995) from Duquesne University, and her PhD in Pharmaceutical Sciences from the University of Wisconsin at Madison (2001). She has been involved in the development of various lyophilized products for biologics including high concentration formulations, formulations of pegylated protein, protein-lipid complexes and conjugated vaccines during her eight plus years of industrial experience.

Satish Singh, Ph.D. is a Research Fellow, Biotherapeutic pharmaceutical science, Global Biologics at the Pfizer Corporation (formerly Pharmacia, Pharmacia & Upjohn. His responsibilities include leading formulation, process and product development activities for biologics, including vaccines, in the organization. He has 20 years experience in the industry in product development activities ranging from oral dosage forms to ophthalmics and parenterals, encompassing small molecules and biologics. He has published more than 20 articles with emphasis on the colloidal and physical chemistry of macromolecules, and holds three issued patents. His interests are in the use of physical chemistry tools for the understanding of formulation characteristics for biologics, as well as their impact on immunogenicity. He has recently been involved in leading a group of industry scientists to examine the concerns around protein-based subvisible particulates. Satish obtained his B.Tech from the Indian Institute of Technology, New Delhi, and an MS and PhD in Chemical Engineering from Kansas State University.

To contact the author please email her directly at: [email protected]