Mycological Assessment of Pharmaceutical Grade Raw Materials

Introduction

It has been estimated that around half of the fungi found in the environment could cause infections in people (mycosis).¹ For this reason, mycoremediation remains an essential part of any pharmaceutical manufacturer’s environmental control program.² A sometimes-overlooked area in the fungal control strategy is raw materials (used as active ingredients, adjuvants, and excipients).³ This is a concern beyond the Microbial Limits Test, in terms of the sometime presence of certain species of fungi that lead to extant mycotoxins. The primary risk is to the herbal products used to manufacture herbal drugs, including medicinal cannabis products. This is a major area of economic activity; the U.S. FDA estimates that some 20,000 different plant species are used in herbal remedies each year.⁴ These products are prone to infestation by microorganisms, especially molds as they are organic in nature.⁵

Mycotoxins are metabolic products of filamentous fungi, characteristic of each species, and they are for the most part heat stable. There are more than 400 different mycotoxins identified to date.⁶ Depending upon the type and the level some mycotoxins are harmful - toxic peptidic toxins. These harmful mycotoxins can be of acute toxicity, causing damage to liver, kidneys, nervous system, skin, mucous membranes, and immune systems. In some cases, chronic toxicity may provoke cancer, may cause congenital abnormalities and malformations in the embryo. The UN Food and Agriculture Organization estimates that 25% of the world production of foodstuffs is contaminated with mycotoxins.⁷ For food for human consumption, a limit of not more than 4 ng.g-1 limit of the specific mycotoxin - aflatoxin - is generally accepted globally.⁸ However, levels vary globally in relation to foodstuffs, with limits in European being lower than those permitted in the U.S.

In terms of risk, this article looks at the types of raw materials that are theoretically under a greater risk of mycotoxin contamination than others. This is in the context of some of the primary mycotoxin producing fungi being the types of fungi with an association with pharmaceutical ingredients and storage or manufacturing environment: species of Aspergillus, Penicillium, and Fusarium. The extent that mycotoxin control matters largely depends on the material and the way the material is stored and handled. While this article makes recommendations for material supplier controls, any risk to a manufacturing process will be product specific (formulation, route of administration etc.) and process specific (in terms of removal steps, ingredients, heating etc.). However, to varying levels the constituent starting materials will affect the quality of the finished product.⁹

Mycotoxins

Many fungi produce biologically active compounds, several of which are toxic to animals or plants and are therefore called mycotoxins.¹⁰ Mycotoxins are produced by growing fungi and their exact purpose is unclear, although host immobilization appears to be one probable reason. The presence of mycotoxins in food or herbal remedies can lead to human disease (mycotoxicosis). The symptoms of mycotoxicosis depend on the type of mycotoxin; the concentration and length of exposure; as well as age, health, and sex of the exposed individual.¹¹ One mold species may produce many different mycotoxins, and several species may produce the same mycotoxin. Many mycotoxins are of little concern because they are either produced by fungi that are rarely encountered in foods or feeds or the concentrations required for toxicity are not encountered. With other mycotoxins, toxicity depends on the route of administration (i.e., what might be toxic by injection is not by ingestion due to chemical breakdown by stomach acid or due to insolubility). Of relevance to pharmaceutical raw materials, some common types of microscopic fungi can produce mycotoxins that can directly harm people, such as Aspergillus spp. and some species of Penicillium spp.¹² In particular, the Penicillia feature among the most common types of fungi isolated from the environment. Of the approximately 150 species recognized, some are frequently implicated in the deterioration of food products, where they may elaborate mycotoxins.¹³ Another risk factor derives from the excessive inhalation of airborne mycotoxins and the connection with allergies and respiratory illnesses.¹⁴ Fungi of specific concern to pharmaceuticals are presented in Table 1.

Given the variation of only some species producing mycotoxins, species identification can be an important control factor and the services of an experienced mycologist may be required. However, the presence of mycotoxins is not necessarily connected to viability; so, absence of fungi from the Microbial Limits Test does not necessarily mean absence of mycotoxins, should certain raw materials be purchased from suppliers of lower quality standards. The time of testing is also an important consideration, since contamination can occur during storage.

Of greater concern are aflatoxins from Aspergillus (triggering the lung disease aflatoxicosis). Aflatoxins in the groups B1, B2, G1, and G2 are of particular concern,¹⁶ as such aflatoxins are potent carcinogens.¹⁷ Aflatoxin B1 is considered the most toxic aflatoxin (produced by A. flavus and A. parasiticus). Also, of interest are ochratoxin A, which is a toxin produced by different Aspergillus and Penicillium species.¹⁸ This is one of the most-abundant food-contaminating mycotoxins (as well as being a contaminant of water-damaged areas and of heating ducts).^19,20 Deoxynivalenol is a mycotoxin associated with Fusarium species (primarily F. graminearum). These fungi produce the largest group of mycotoxins (trichothecenes) although the association is primarily with corn and animal feedstuffs.²¹

Raw Materials at Risk

Risk of mycotoxin will relate to the raw material and its point of origin or to the storage conditions of the material. For example, the common habitat of Aspergillus is in soil, decaying vegetation, hay, and grains undergoing microbiological deterioration. However, the fungus can invade almost all forms of organic substrates under favorable conditions. Favorable conditions include high moisture content (at least 7%) and higher temperature.²²

Raw materials at risk are:

• Herbal raw materials, where fungi grow naturally in association with.
• Vegetative raw materials such as vegetable oils.
• Agricultural raw materials, including soil derived materials like calcinated earth (especially in areas where peanut seeds are present)
• Materials of animal origin, where mycotoxins can accumulate in animal tissues, rendering the affected organs/tissues unfit for use as a source of starting material for the production of animal-derived drug ingredients.

This means that only a few materials are contaminated and hence quality control is only required for critical products or products from sources where there is potential concern (but no other alternative). Yet for those materials considered at risk, contamination can be extensive.

For example, in one study researchers in India looked at six common herbal raw materials and found all samples to be contaminated, with a total of 41 fungal species belonging to 16 genera were isolated and identified. Acorus calamus Linn., Cassia angustifolia Vahl., Centella asiatica (Linn.) Urban, Myristica fragrans Houtt., Tinospora cardifolia (Wild) Miers and Withania somnifera (Linn.) Dunal. Of the fungi, Aspergillus and Penicillium were more predominant. A. niger was found to be most frequently occurring fungi followed by A. flavus.²³

While specific materials present a risk, the extent to which the mycotoxin hazard is removed through processing will be product specific and only human biomonitoring studies can provide realistic data on internal exposure at individual level, allowing an assessment of the determinants of exposure to these contaminants.

Raw Material Control

The quality of medicinal products is laid down in the European Pharmacopoeia (Ph.Eur.) and the United States Pharmacopeia. The Pharmacopoeias describe the parameters relevant for the quality of herbal drugs and herbal drug preparations in monographs. The Pharmacopoeias define the quality parameters based on the available scientific knowledge at the time of publication, which means that other, more recent, scientific literature may need to be referred to. Pharmacopeias focus on quality parameters of the starting materials, not on finished products. Where testing is required this will primarily be for aflatoxin and ochratoxin A (based on a risk assessment). With test methods, as described below, mycotoxins are readily soluble in alcoholic solvents and an analysis of the extracts can be performed.

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As recommended in ICH Q7, suppliers of raw materials from specific risk groups should undertake a risk assessment and determine the excipient quality, labelling and delivery requirements of the customer, in relation to mycotoxin levels.²⁴ A suitable risk assessment tool is HACCP (Hazard Analysis and Critical Control Points).²⁵ The pharmaceutical company purchasing the materials should be confident that such assessment has taken place and that appropriate testing (as discussed below) has been carried out.

Where a risk is suspected, evidence should be provided that the supplier has developed strategies to avoid contamination in the source (such as during drying or storage). With materials of plant origin, controlled indoor conditions offer better protection than outside environment where open agricultural fields are exposed to phytopathogenic and mycotoxigenic fungi.²⁶ For materials at risk, the manufacturer of the material must practice Good Agricultural Practice (GACP). Examples include.^27,28

• Preventing molds from growing in drug ingredients and when feasible and valuable remove all molds that may contaminate such ingredients.
• Limiting bird and insect damage, since fungi tend to invade damaged kernels more easily than intact ones.
• Harvesting as soon as possible. Many fungi grow readily under damp conditions.
• Adequate drying and storage to prevent fungal growth and mycotoxin production post-harvest. Being suitably dry lowers the water activity required for common fungal growth. Heating can also eliminate fungal growth, with few common fungi surviving above 45°C. The D-value can be assessed for the most common types of fungi, should heating be possible in terms of not damaging the material. For example, the D60 value of Aspergillus is around 1 minute.²⁹
• Ensuring conditions remain anaerobic to limit fungal growth and mycotoxin contamination. Fungi cannot grow under truly anaerobic conditions.
• Segregating herbs intended for pharmaceuticals away from other agriculture that might be susceptible to fungal infestation (such as in adjacent fields), to minimize disease spread.

Aside from GACP, there are different methods that suppliers can use to address mycotoxin risks. These can be divided into: Removal of mycotoxins from raw materials and/or finished products, transformation (modification of the chemical structure of the molecule), detoxification (transformation which reduced the toxicity), and decontamination (removal or detoxification/inactivation).³⁰

As an example, for particular ‘at risk’ products, such as vegetable oils (including those derived from arachis seeds), the manufacturer may need to undertake the process of refining. The objective of refining is to remove impurities and contaminants of the oil with the least possible damage to the triglycerides and with minimal loss of oil. Another method involves removing mycotoxin by using binding agents such as montmorillonite or bentonite clay. These substances can adsorb mycotoxins. Alternatively, enzymes (such as esterase), some species of yeasts or bacterial strains, can absorb mycotoxins. Techniques for physical separation can also be deployed, such as washing, milling, nixtamalization (such as soaking or cooking in an alkaline solution), heat-treatment, and extraction with solvents. To destroy the source of mycotoxins, irradiation methods can be effective against mold growth and toxin production.³¹

Laboratory Testing

Given there is data of aflatoxin and ochratoxin A residues existing in herbal origin materials, for particular at-risk material groups or processes testing may need to be performed. As the knowledge on the occurrence of different mycotoxins in herbal materials is limited, the compendia focus on the detection of the most harmful and prevalent: Aflatoxin (especially B1) and ochratoxin A. For example: USP general chapter <561> Articles of Botanical Origin: Test for Aflatoxins, where the limits in the USP are not more than 20 parts per billion (ppb) for the total of aflatoxins B1, B2, G1, and G2 combined and not more than 5 ppb of aflatoxin B1. The equivalent chapter in Europe is Ph. Eur. 2.8.18 (the methods described have been qualified as suitable for testing devil’s claw root, ginger and senna pods; for testing other products method suitability must be demonstrated). The European limit is not more than 2μg/kg of aflatoxin B1; plus, a limit of 4μg/kg for the sum of aflatoxins B1, B2, G1 and G2 (where required). There is an additional European chapter on ochratoxin A, which is Ph. Eur. 2.8.22 (the methods have been qualified as suitable for the examination of liquorice extract and liquorice root, and against other products method suitability must be demonstrated). The limits for ochratoxin A vary by product and are higher than for aflatoxins.

Mycotoxins are difficult to test for and as indicated earlier, harmful at the parts per billion (ppb) level; plus, the sample preparation is relatively complex. Several sampling and analytical methods including Thin-Layer Chromatography (TLC), High-Performance Liquid Chromatography (HPLC), mass spectrometry, and Enzyme-Linked Immunosorbent Assay (ELISA), have been used to test for aflatoxin B1 contamination.³² After a matrix has been extracted from a sample and filtered, and then tested via chromatographic methods, with detection limits around 2-5 ppb, or by ELISA antibody kits with the detection limits down to single parts per trillion. An alternative to ELISA to measure hazardous aflatoxins, such as B1, is the fluorescence polarization immunoassay (FPIA).³³ Ideally, such testing, given its specialist nature, will be undertaken by the raw material supplier or by a certified contract test facility.

Summary

Mycotoxins will present an impurity risk to some raw materials and hence to some types of pharmaceutical product (primarily those of an herbal basis). To minimize the risk, approved suppliers providing materials of pharmacopeial grade should be selected. Pharmaceutical companies should assess which of their incoming materials is at a potential hazard and ensure that the audit of the supplier considers the supplier’s own risk-based approach to minimizing mycotoxin contamination and how the supplier has assessed the success of these measures, such as through periodic assay. The article has considered testing methods, which should be conducted by the supplier. Despite the variety, all analytical methods require solvent extraction of the mycotoxin of interest from the matrix, followed by key analytical steps. While testing is important, the most important activity is lowering the chance of contamination occurring in the first place, either through harvesting, storage, or transport.

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