A Novel Tandem-Tag Purification Strategy for Challenging Disordered Proteins.

Intrinsically disordered proteins (IDPs) lack well-defined 3D structures and can only be described as ensembles of different conformations. This high degree of flexibility allows them to interact promiscuously and makes them capable of fulfilling unique and versatile regulatory roles in cellular processes. These functional benefits make IDPs widespread in nature, existing in every living organism from bacteria and fungi to plants and animals. Due to their open and exposed structural state, IDPs are much more prone to proteolytic degradation than their globular counterparts. Therefore, the purification of recombinant IDPs requires extra care and caution, such as maintaining low temperature throughout the purification, the use of protease inhibitor cocktails and fast workflow. Even so, in the case of long IDP targets, the appearance of truncated by-products often seems unavoidable. The separation of these unwanted proteins can be very challenging due to their similarity to the parent target protein. Here, we describe a tandem-tag purification method that offers a remedy to this problem. It contains only common affinity-chromatography steps (HisTrap and Heparin) to ensure low cost, easy access and scaling-up for possible industrial use. The effectiveness of the method is demonstrated with four examples, Tau-441 and two of its fragments and the transactivation domain (AF1) of androgen receptor.

The occurrence of putative cognitive enhancing research peptides in seized pharmaceutical preparations: An incentive for controlling agencies to prepare for future encounters of the kind.

At the end of 2017 and 2018 two different unknown suspicious preparations were encountered and were subjected to a plethora of different analyses in order to identify, if present, any bioactive compound. It turned out that these samples contained the assumedly cognitive enhancing research peptides Selank and Semax, which, to our knowledge, have not completed any clinical trials. Moreover, an online search, excluding the dark web, demonstrated that these kinds of nootropic research peptides are freely available either as lyophilized powder for injection purposes or are present in nasal sprays. It stands to reason that controlling laboratories need to anticipate the uprising of these types of potentially dangerous molecules and must therefore be able to correctly identify these compounds. Therefore, these findings served as an incentive to develop a novel combined liquid chromatography tandem mass spectroscopy (LC-MS/MS) methodology, applicable to both hydrophilic or more hydrophobic peptides, which was utilized to analyze a total of 10 putative cognitive enhancing polypeptides, with variable biochemical characteristics, that are currently being sold online. The screening rationale, complying to the recommendation paper of the General European Official Medicines Control Laboratory (OMCL) network on the interpretation of screening results for unknown peptides by mass spectrometry, was also validated in different matrices as required by ISO 17025.

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.

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 tomorrow's 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.

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.

Assessment of dietary intake of 10 intense sweeteners by the Italian population.

The aim of the present study was to monitor the consumption of foods containing intense sweeteners present on the Italian food market and to investigate whether the Italian general population (aged >3-65+) was at risk for exceeding the Acceptable Daily Intake (ADI) of 10 intense sweeteners. A food label survey was performed in Rome (Italy), using market share data to identify the brands more representative of the market. A sample of 326 foods (table-top sweeteners included), beverages and food supplements containing intense sweeteners was collected and analyzed in order to establish the concentration levels. Intense sweeteners were only found in foods belonging to 8 sugar-free food categories out of 37 regulated. The dietary exposure was estimated using the tiered approach. Food consumption data from the last Italian national survey (INRAN-SCAI 2005-06) were combined with Maximum Levels at Tier 2, and with the actual concentration of sweeteners in the collected food products at Tier 3. The estimated exposure among consumers of sweeteners in Italy was well below the ADIs, in both tiers; non-alcoholic beverages, table-top sweeteners and food supplements were main contributors to exposure.

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.

Low-calorie sweeteners in food and food supplements on the Italian market.

This study determines the occurrence and concentration levels of artificial low-calorie sweeteners (LCSs) in food and food supplements on the Italian market. The analysed sample set (290 samples) was representative of the Italian market and comprised of beverages, jams, ketchups, confectionery, dairy products, table-top sweeteners and food supplements. All samples were analysed via UPLC-MS/MS. The method was in-house validated for the analysis of seven LCSs (aspartame, acesulfame-K, saccharin, sucralose, cyclamate, neotame and neohesperidin dihydrochalcone) in food and for five LCSs (aspartame, acesulfame-K, saccharin, cyclamate and sucralose) in food supplements. Except for cyclamate in one beverage which exceeded the maximum level (ML) with 13%, all concentrations measured in food were around or below the ML. In food supplements, 40 of the 52 samples (77%) were found to be above the ML, with exceedances of up to 200% of the ML.

Analysis of illegal peptide biopharmaceuticals frequently encountered by controlling agencies.

Recent advances in genomics, recombinant expression technologies and peptide synthesis have led to an increased development of protein and peptide therapeutics. Unfortunately this goes hand in hand with a growing market of counterfeit and illegal biopharmaceuticals, including substances that are still under pre-clinical and clinical development. These counterfeit and illegal protein and peptide substances could imply severe health threats as has been demonstrated by numerous case reports. The Belgian Federal Agency for Medicines and Health Products (FAMHP) and customs are striving, together with their global counterparts, to curtail the trafficking and distributions of these substances. At their request, suspected protein and peptide preparations are analysed in our Official Medicines Control Laboratory (OMCL). It stands to reason that a general screening method would be beneficiary in the battle against counterfeit and illegal peptide drugs. In this paper we present such general screening method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the identification of counterfeit and illegal injectable peptide preparations, extended with a subsequent quantification method using ultra-high performance liquid chromatography with diode array detection (UHPLC-DAD). The screening method, taking only 30 min, is able to selectively detect 25 different peptides and incorporates the proposed minimum of five identification points (IP) as has been recommended for sports drug testing applications. The group of peptides represent substances which have already been detected in illegal and counterfeit products seized by different European countries as well as some biopharmaceutical peptides which have not been confiscated yet by the controlling agencies, but are already being used according to the many internet users forums. Additionally, we also show that when applying the same LC gradient, it is also possible to quantify these peptides without the need for derivatization or the use of expensive labelled peptides. This quantification method was successfully validated for a representative subset of 10 different peptides by using the "total error" approach in accordance with the validation requirements of ISO-17025.