Celine Vanhee

Identification and characterization of peptide drugs in unknown pharmaceutical preparations seized by the Belgian authorities: case report on AOD9604

Counterfeit medicines, imitations, and substandard medicinesare a growing problem worldwide. The problem is situatedboth in developing countries as in industrialized regions. Inthe developing countries, the problem concerns the wholemedicine supply chain, especially essential medicines likeantibiotics,

Analysis of illegal peptide drugs via HILIC-DAD-MS

Biopharmaceuticals have established themselves as highly efficient medicines, and are still one of the fastest growing parts of the health-product industry. Unfortunately, the introduction of these promising new drugs went hand in hand with the creation of a black market for illegal and counterfeit biotechnology drugs. Particularly popular are the lyophilised peptides with a molecular weight of less than 5kDa. Most of them are meant for subcutaneous injection and are easily accessible via the internet. In recent years, different methods based on reversed phase liquid chromatography have been developed to detect and quantify these peptides. The emerging of more polar peptides however requires the introduction of other separation techniques. Therefore, we set out to develop and validate an analytical method based on hydrophilic interaction liquid chromatography (HILIC) to identify and quantify the most frequently encountered illegal peptides on the European market. For this objective, five different HILIC columns were selected and screened for their chromatographic performance. Among those columns, the ZIC HILIC column showed the best performance under the tested screening conditions in terms of resolution and symmetry factor for the targeted peptide set. Hence, the operational conditions were further optimised for the identification of illegal preparations via mass spectrometry (MS) and quantification via UV. Validation was performed via accuracy profiles based on the ISO 17025 guideline. The obtained validated HILIC-method allows for the detection and quantification of the most frequently encountered illegal peptides on the internet in a total run time of 35min including post gradient equilibration and online cleaning step. Combined with a previously developed RPLC-method, the ZIC HILIC system allows for the detection and quantification of a wide spectrum of illicit peptide drugs available on the internet. Furthermore, the developed method could also be envisaged for the detection of new emerging polar peptide drugs.

Identification of the small research tetra peptide Epitalon, assumed to be a potential treatment for cancer, old age and Retinitis Pigmentosa in two illegal pharmaceutical preparations.

SFFC (Spurious/falsely-labelled/falsified/counterfeit) medicines are a growing problem worldwide. While the problem in the developing countries concerns principally essential medicines like antibiotics, HIV medication, and anti-malaria products, the problems in the industrialized world concerns mostly drugs for erectile dysfunction, anabolic hormones, and slimming products. Although occasionally counterfeited antibiotics, insulin, and interferon are also intercepted by the customs or controlling agencies. Moreover, there is also a growing market for non-approved drugs, including peptides and proteins, which are being made available to the public before entering or completing clinical trials. Global actions are taken against criminal entities to curtail the trafficking and distribution of these substances. The growing threat of these products in Europe is mainly due to the extension of the Internet, where about 50% of the medicines sold through Internet sites, disclosing their identity, are estimated to belong to this category. In order to protect the costumer against such malice, a European directive was put in place in 2011. A new logo for online purchases has to be implemented by member states by July 2015. This logo will allow patients and consumers to identify authorized online pharmacies providing authentic, authorized medicines. At the request of the Federal Agency for Medicines and Health Products (FAMHP), unknown pharmaceutical preparations suspected to contain SFFC drugs, including protein and peptide drugs, are regularly subjected to analysis in the laboratory. Although almost all of the proteins and peptides analyzed cover potential doping agents, the SFFC version of insulin, Botulinum toxin, human interferon 1 α, nasal sprays containing the neuropeptide oxytocin, human serum albumin, and the potential toxic skin tanning peptide Melanotan II, were also found recently. In the last year, we have also received two different suspected illegal samples that contained a substance with a mass of approximately 390 Da, corresponding to a peptide with sequence AEDG. The researchers who generated this peptide, termed Epitalon, claim

A simple dilute and shoot methodology for the identification and quantification of illegal insulin

The occurrence of illegal medicines is a well-established global problem and concerns mostly small molecules. However, due to the advances in genomics and recombinant expression technologies there is an increased development of polypeptide therapeutics. Insulin is one of the best known polypeptide drug, and illegal versions of this medicine led to lethal incidents in the past. Therefore, it is crucial for the public health sector to develop reliable, efficient, cheap, unbiased and easily applicable active pharmaceutical ingredient (API) identification and quantification strategies for routine analysis of suspected illegal insulins. Here we demonstrate that our combined label-free full scan approach is not only able to distinguish between all those different versions of insulin and the insulins originating from different species, but also able to chromatographically separate human insulin and insulin lispro in conditions that are compatible with mass spectrometry (MS). Additionally, we were also able to selectively quantify the different insulins, including human insulin and insulin lispro according to the validation criteria, put forward by the United Nations (UN), for the analysis of seized illicit drugs. The proposed identification and quantification method is currently being used in our official medicines control laboratory to analyze insulins retrieved from the illegal market.

Identification of epidermal growth factor (EGF), in an unknown pharmaceutical preparation suspected to contain insulin like growth factor 1 (IGF-1).

The last 30years are marked by a tremendous evolution in the comprehension of human physiology. Although many physiological processes remain enigmatic, our increased understanding has led to a considerable development of medicines. A small, but yet a more progressively important sub fraction of this drug market consists of recombinant proteins and peptides. Although in most western countries a prescription is required for the injection of these compounds, some of them can easily be acquired from illicit internet pharmacies whether or not disguised as ‘research companies’. Some of these drugs can not only be used to cure ourselves but also to improve our sports performance, deepen our tan, and strive to the ideals imposed by society. Reports originating from controlling agencies residing in Germany, the UK, Denmark, Norway, Italy, the USA, Australia, and Belgium show that monoclonal anti‐cancer antibodies, insulins, growth hormones, human chorionic gonadotropin, Clostridium botulinum toxin type A, potential doping peptides, putative anti‐obesity drugs, skin tanning peptides, neuropeptides and a putative anti‐cancer polypeptide were identified in seized samples. The danger of these illegal polypeptides resides in the fact that these polypeptide products are not produced under controlled environment and therefore may contain the wrong ingredient, the wrong dosage, bacteria, viruses, heavy metals, etc., which could result in severe health issues. In some cases these illegal acts had a deadly outcome. Furthermore, for many of these polypeptide drugs, there is a limited knowledge of their effects and/or side‐effects (long and short term) hence clinical studies have not been terminated or even worse were given a negative advice due to overall potential adverse health effects. Global actions are on‐going to protect the sometimes unaware public from these malignant activities. In Belgium, the Federal Agency for Medicines and Health Products (FAMHP) is responsible for this task and upon their request unknown pharmaceutical preparations suspected to contain illegal drugs are regularly subject to analysis in our official medicines control laboratory (OMCL). At the end of 2014, an unknown pharmaceutical preparation, claimed to contain insulin like growth factor 1 (IGF‐1) was submitted for analysis by immunoblotting against IGF‐1 and analogues, prior to liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Based on the interpretation of the western blot and the LC‐MS data we were not able to identify this substance as being any form of insulin like growth factor (IGF), including IGF‐1, IGF‐2 or synthetic analogues potentially used as doping agent. Further analysis by LC‐MS/MS and subsequent peptide sequencing showed that the substance present in the sample corresponds to epidermal growth factor (EGF). EGF plays a major role in the regulation of cell growth, proliferation and differentiation and is therefore frequently used in cell culture. This polypeptide, consisting of 53 amino acids, with sequence NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR and 3 intramolecular disulfide bonds has an average Molecular weight of 6215.9Da. This polypeptide is known to interact with the EGF‐receptor (EGFR) and can promote tumour cell motility and invasion. Therefore the blocking of EGF binding to the EGFR and further downstream signalling pathway is currently marked as a target for anti‐cancer therapy for certain cancers (reviewed in Yewale et al.). Indeed, a vaccine targeting EGF, named CimaVax‐EGF, results in the production of antibodies directed against EGF and this vaccine is being used as cancer therapy against non‐small‐cell lung carcinoma in Cuba. The CimaVax vaccine consists of recombinant EGF chemically conjugated to the protein P64K from Neisseria meningitides. It induces the production of antibodies directed against EGF and results in the prevention of EGF to attach to EGFR and thus inhibiting the signal that tells cancer cells to grow and divide. Nowadays, the product is undergoing further trials for possible licensing in Europe and the United States. Although no injectable legal medicine is available on the market, the product is used in the cosmetic industry. It is thought that the topical application of EGF would enhance wound healing and stimulate skin rejuvenation. Taken together, we believe that the purchaser was unaware of the presence of EGF in the sample. This case of potential dangerous wrong labelling clearly demonstrates the peril of purchasing potential doping agents via the black market.

Impurity profiling of the most frequently encountered falsified polypeptide drugs on the Belgian market

Advances in biotechnology and the chemical synthesis of peptides have made biopharmaceuticals and synthetic peptide drugs viable pharmaceutical compounds today and an important source for tomorrows drugs and therapies. Unfortunately, also falsifications and counterfeit versions of these powerful and promising drugs are offered illegally via the internet. Since these falsified preparations are produced outside the legally required quality systems, end-users have no guarantee regarding the efficacy and safety of these products. Although falsified samples of biotherapeutics were already analysed, looking at a specific aspect of their quality or identity, no systematic studies have been performed regarding the presence of different impurities or possible contaminations. Therefore, in order to obtain a better understanding of the potential health risks related to the usage of falsified polypeptide drugs we performed a systematic screening of the ten most frequently encountered falsified peptide drugs on the Belgian market acquired from three different suspected illegal internet pharmacies. The screening incorporated the analysis of the active pharmaceutical ingredient (API), API-related impurities, small molecule contaminants (defined as organic small molecules not belonging to the other categories), elemental impurities and residual solvents. This comprehensive study showed that these type of falsified drugs not only have a high variation in amount of drugs per unit and a low purity (ranging between 5% and 75% for cysteine containing peptides), but also contained the known toxic class one elemental impurities arsenic (As) and lead (Pb). One sample was contaminated with Pb while multiple samples were found with concentrations up to ten times the ICH toxicity limit for parenteral drugs. Subsequent speciation of As confirmed the elevated concentrations for As and demonstrated that all As was present in the more toxic inorganic form. Together with the (sometimes) high amount of peptide impurities and the inherent dangers associated with the use of unauthorized peptide drugs (such as doping peptides or preclinical drugs) this study confirms the reported potential health risks patients/users take when resorting to falsified peptide drugs. Moreover, the presence of the carcinogen As and the known accumulation in human tissues of Pb raises questions about potential sub-acute to chronic toxicity due to the long term administration of these falsified peptide drugs.

A high crosslinking grade of hyaluronic acid found in a dermal filler causing adverse effects

Graphical abstract Figure. No caption available. HighlightsA method is developed for the routine analysis of dermal filler products.The method is used to identify modified hyaluronic acid and to quantify the amount of modifications and crosslinks.A product previously retrieved from the market was found to contain a high modification and crosslinking grade, which may be the reason for the high risk for adverse effects of this product.The product retrieved from the market appeared to be unreliable in its composition, regarding the modification grade and presence of a local anesthetic.There is a clear need for analytical methods to test medical devices on a routine basis as shown by the presence of suspicious and substandard products available on the market. ABSTRACT Facial treatments with dermal fillers for medical or esthetic purposes occasionally give rise to adverse effects, ranging from temporary effects such as reddening of the skin, to long term effects such as hardening of tissue. There appears to be a relationship between the lifetime of the filler product and the risk for adverse effects. The lifetime of hyaluronic acid‐based fillers is dependent on the presence and amount of crosslinking agents such as 1,4‐butanediol diglycidyl ether (BDDE). It would therefore make sense to establish methodology to analyze the crosslinking grade of HA‐based filler products on a routine basis. To this end, an analytical method was developed and validated to identify HA‐BDDE‐based fillers and to quantify their modification and crosslinking grade. The method was subsequently applied to products from the legal supply chain and the illegal market. It was found that the product Hyacorp H 1000, previously taken from the market, indeed contains a high modification grade and crosslinking grade, as was the assumed reason for the increased risk for adverse effects of this product. However, it was also shown that the Hyacorp products are highly unreliable in relation to their product composition in general. In this study, authentic products could not be distinguished from the illegal market products based on their modification and crosslinking grade.

Falsification of biotechnology drugs: current dangers and/or future disasters?

Falsified medical products have become a global threat since they were first mentioned to the general public at the conference of experts on the rational use of drugs organized by the world health organization (WHO) in 1985. Today, official estimates of the annual death toll due to falsified medical products range between two hundred thousand and one million. Although the extent of this global problem is the most significant in the developing world, an increasing number of reports have demonstrated the presence of a substantial (black) market for falsified medical products in the developed world. In recent years, also biotechnology drugs (synthetic peptide drugs and protein drugs) have been reported to be prone for falsifications. Next to the traditional doping related substances and image-enhancing polypeptides (e.g., human growth hormone, melanotan II) also essential medicines such as insulin, oxytocin and monoclonal antibodies have been falsified. The danger regarding the use of these falsified polypeptide drugs lies in the fact that end-users have no guarantee of the safety and efficacy of these preparations. Multiple reports have namely described the presence of the wrong active pharmaceutical ingredient (API), the wrong dosage or the absence of the API. Additionally, adverse health effects have been reported in the past due to toxic contaminations and product or process related impurities. Moreover, also unauthorized polypeptides or polypeptides which failed clinical trials or are still subject of clinical or pre-clinical assessments have been found in seizures of regulatory agencies. It stands to reason that regulatory agencies and analytical laboratories handling falsified biotechnology drugs have stepped up efforts to counter these grievous practices. The analysis of these falsified polypeptides and putative impurities is however not always straightforward. Often (bio)analytical laboratories have to resort to a combination of electrophoretic techniques, immunological assays and mass spectrometry based approaches to merely identify the content of seized samples. In addition, the difference in size (peptide vs proteins vs monoclonal antibodies), complexity (e.g., isoforms, glycosylations) and different synthesis techniques (chemical synthesis, recombinant expression, native protein isolation) result in a wide range of putative health risks. This review therefore aims to provide a brief overview of the genuine biotherapeutics present on the market and their quality prerequisites. Next, we describe the identification strategy utilised by our lab to identify the API in falsified biotherapeutics, followed by a discussion of the putative hazards due to impurities and contaminations that were found or could be encountered in falsified biotherapeutics. Finally, we terminate with an educational prediction of what may happen in the future and possible ways to counteract putative future disasters.

Identification and Quantification Methodology for the Analysis of Suspected Illegal Dietary Supplements: Reference Standard or no Reference Standard, that the Question

The consumption of food supplements has become a billion dollar business that has proliferated across the globe. Consequently this also resulted in the increase of adulterations by the illegal addition of medicinal substances or their analogues. The vast majority of the pharmaceutical substances encountered in those samples belong to the three ‘classical’ categories, including sexual enhancers (e.g. PDE-5 inhibitors and flibanserine), weight loss enhancers occasionally accompanied with an anti-depressant (e.g. sibutramine, phenolphthalein, sibutramine and fluoxetine or venlafaxine) and sports performance enhancers (e.g. selective androgen receptors (SARMs) and anabolic-androgenic steroids (AAS)). Recently also chemicals belonging to the class of pharmaceutical cognition enhancers or nootropics are gaining popularity amongst the general public. While for many of the classical categories reference standards are available to unambiguously confirm the identity of a pharmaceutical product, for several molecules, including some nootropics, no certified reference material is available. Hereby, with the example of an adrafinil containing food supplements, we show how, by utilising multiple methodologies, we were able to come to identification and subsequent quantification when no certified reference standard was available. Furthermore, in 2017 already several food supplements analysed by our lab were positive for the presence of nootropic substances, indicating that these compounds are really gaining popularity amongst the general public.