Characterisation of modified pharmaceutical proteins: the somatropin case

Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous human growth hormone, somatotropin [1]. This protein is clinically used in children and adults with inadequate endogenous growth hormone to stimulate a normal bone and muscle growth. In addition, somatropin is currently being investigated for the diagnosis and radiotherapy of certain hormonal cancers. The modification of the protein with a chelating agent like NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein. The NOTA unit is selectively introduced on a lysine side chain. This yields 9 possible labelling sites for somatropin: 38, 41, 70, 115, 140, 145, 158, 168 and 172. As site-specific labelling is necessary to avoid active region interactions, characterisation of the chelate-modified somatropin is indispensable. Therefore, we have applied an enzymatic digestion procedure using trypsin, chymotrypsin and Staphylococcus aureus V-8 proteases. The resulting peptides were then monitored using HPLC-MSn, allowing the investigation of the exact position of amino acid modifications

Faecal microbiota transplantation : a regulatory hurdle?

During faecal microbiota transplantation, stool from a healthy donor is transplanted to treat a variety of dysbiosis-associated gut diseases. Competent authorities are faced with the challenge to provide adequate regulation. Currently, regulatory harmonization is completely lacking and authorities apply non-existing to most stringent requirements. A regulatory approach for faecal microbiota transplantation could be inserting faecal microbiota transplantation in the gene-, cell-and tissue regulations, including the hospital exemption system in the European Advanced Therapy Medicinal Products regulation, providing a pragmatic and efficacy-risk balanced approach and granting all patients as a matter of principle access to this therapy

Exploring the Brainpeps database

Since the discovery that peptides can cross the blood-brain barrier (BBB), doors have been opened to new therapeutics for CNS diseases and pain management. Recently, we have constructed the Brainpeps database (brainpeps.ugent.be) to give an overview of the available BBB transport data of peptides, which are scattered in the literature [1]. One possible application of the Brainpeps database is the study of structure-property relationships (QSPRs). Before peptides can be used as drugs, their impurity profile needs to be examined as part of the International Conference on Harmonization (ICH) risk assessment of peptide drugs. Compared to small molecules, no in-silico predictive programs are available for toxicity screening of the different peptide impurities towards passing the BBB. To predict the BBB-behaviour of peptides as well as their impurities, we explored the Brainpeps database. During this presentation, the first results of the modelling experiments are presented. Our starting hypothesis is that the interactions of peptides at the blood-brain barrier are comparable with those of peptides in HPLC systems. Therefore, we determined the retention characteristics on different fused-core HPLC systems of a set of model peptides selected from the Brainpeps database and explored the relationship between the chromatographic characteristics and their BBB-influx properties [2]. In conclusion, using the Brainpeps database and experimental HPLC data, a first step towards in-silico profiling of peptides, including their impurities, at the blood-brain barrier level is taken. More chromatographic analyses of BBB peptides and harmonization on testing the BBB transport of peptides are future challenges to validate and unify this model. References [1] Van Dorpe S., Bronselaer A., Nielandt J., Stalmans S., Wynendaele E., Audenaert K., Van de Wiele C., Burvenich C., Peremans K., Hsuchou H., De Tré G., De Spiegeleer B. Brainpeps: the blood-brain barrier peptide database. Brain Struct Funct (2012), DOI: 10.1007/s00429-011-0375-0. [2] D’Hondt M., Van Dorpe S., Gevaert B., Wynendaele E., Stalmans S., Peremans K., Burvenich C., De Spiegeleer B. Fused-core RP-HPLC modelling of peptides. Journal of Pharmaceutical Analysis (2012), Accepted for publication

Exploring the chemical-functional space of cell-penetrating peptides

Cell penetrating peptides (CPPs) are an increasingly growing part of fundamental and applied peptide research. Using their capacity to cross cell barriers, they have already been successfully applied as carriers for problematic cargos like DNA, (si)RNA, proteins and other peptides, (poly/oligo) saccharides and small molecules. Several hundreds of CPPs, showing different properties and activities, are already reported in the literature. To clarify the different types of actions in cell-penetrating behaviour, a database of more than 200 peptides was build, covering the CPPs described over the last five years and for which quantitative data were available. Seen the wide range of techniques, cell lines, peptide concentrations and other operational parameters used to quantify the cellular uptake, a unified response for cellular uptake was firstly defined based upon a concentration corrected standardized response relative to the concentration corrected response of Penetratin, a well known and characterized CPP. In this way a “meta-analytical” comparison of the cellular uptake of different CPPs is established, which was currently hardly needed. Therefore a chemical space was developed using more than 3000 descriptors, calculated from the optimized 3D-structure of the CPPs. By combining these descriptors and the unified responses, clusters of peptides are obtained from which model CPPs can be rationally selected and QSPRs and mechanisms of action established

Influence of variability in starting materials quality on stability of finished drug products: a quality-by-design factor and response

The use of ill selected excipients in drug formulations can have a significant influence on the overall stability. Therefore, evaluation of chemical and physical excipient compatibility with the API has become a major part in the development of new drug products. Moreover, general and individual limits for excipient impurities have also been set by the Ph. Eur. However, batch to batch variability of these excipient impurities, although still Ph. Eur compliant, can cause significant variability in the stability profile of finished drug product. Recently, large manufacturer and batch to batch variability in hydroperoxide levels was documented in common used pharmaceutical excipients such as povidone, polysorbate 80, PEG 400 and hydroxypropylcellulose [1]. As a result, oxidation sensitive drugs, e.g. raloxifene HCl, can demonstrate inconsistent stability profiles when combined with aforementioned excipients [2]. Another example in which a miconazole-BHT adduct is formed, can be traced back to the petrolatum vehicle, containing BHT, used for topical application [3]. Note that no BHT limits are mentioned in the corresponding Ph. Eur. monograph. We evaluated the short-term storage stability of three triple intrathecal (Triple IT) solution batches under various conditions [4]. The Triple IT solution, containing cytarabine, methotrexate and methylprednisolone (21)-sodium succinate (MPSS), is used in the treatment of leukemia, lymphoma and brain cancers. Hydrolysis of MPSS to methylprednisolone was found to be the predominant degradation reaction. However, different MPSS degradation kinetics were observed. This observation was linked to the use of different batches of MPSS starting material, i.e. Solu-Medrol®, thus providing an inconsistency in the degradation profile. References [1] Wasylaschuk, W.R.; Harmon, P.A.; Wagner, G.; Harman, A.B.; Templeton, A.C.; Xu, H.; Reed, R.A. Evaluation of hydroperoxides in common pharmaceutical excipients (2006). Journal of Pharmaceutical Sciences; 96; 106-116. [2] Hartauer, K.J; Arbuthnot, G.N.; Baertschi, S.W.; Johnson, R.A.; Luke, W.D; Pearson, N.G.; Rickard, E.C.; Tingle, C.A.; Tsang, P.K.S.; Wiens, R.E. Influence of peroxide impurities in povidone and crospovidone on the stability of raloxifene hydrochloride in tablets: identification and control of an oxidative degradation product (2000). Pharmaceutical Development and Technology; 5; 303-310. [3] Zhang, F.; Nunes, M. Structure and generation mechanism of a novel degradation product formed by oxidatively induced coupling of miconazole nitrate with butylated hydroxytoluene in a topical ointment studied by HPLC-ESI-MS and organic synthesis. [4] D’Hondt, M.; Vangheluwe, E.; Van Dorpe, S.; Boonen, J.; Bauters, T.; Pelfrene, B.; Vandenbroucke, J.; Robays, H.; De Spiegeleer, B. Stability of extemporaneously prepared Triple inthrathecal solution of cytarabine, methotrexate and methylprednisolone sodium succinate (in press). American Journal of Health-System Phamacy

Analysis of iodinated quorum sensing peptides by LC-UV/ESI ion trap mass spectrometry

Five different quorum sensing peptides (QSP) were iodinated using different iodination techniques. These iodinated peptides were analyzed using a C-18 reversed phase HPLC system, applying a linear gradient of water and acetonitrile containing 0.1% (m/v) formic acid as mobile phase. Electrospray ionization (ESI) ion trap mass spectrometry was used for the identification of the modified peptides, while semi-quantification was performed using total ion current (TIC) spectra. Non-iodinated peptides and mono- and di-iodinated peptides (NIP, MIP and DIP respectively) were well separated and eluted in that order. Depending on the used iodination method, iodination yields varied from low (2%) to high (57%)

LC-MS characterization and cell-binding properties of chelate modified somatropin

Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous human growth hormone, somatotropin. This protein is clinically used in children and adults with inadequate endogenous growth hormone to stimulate a normal bone and muscle growth. In addition, somatropin is recently being investigated for the diagnosis and radiotherapy of certain hormonal cancers. In some of these cancers, over-expression of the human growth hormone receptor (hGHR) is described. The modification of the protein with a chelating agent like NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein. The NOTA unit is selectively introduced on a lysine side chain. As site-specific labelling is necessary to avoid active region interactions (1-16, 41-68, 103-119 and 167-175), characterization of the chelate-modified somatropin is indispensable. Therefore, we have applied an enzymatic digestion procedure using trypsin, chymotrypsin and a combination of both enzymes. The resulting peptides were then monitored using HPLC-MSn, allowing the investigation of the exact amino acid modifications. The use of a mixture of trypsin and chymotrypsin gave an enhanced information efficiency. Moreover, the intact protein, without enzymatic degradation, was analysed on a protein HPLC column using UV detection for quantification and ESI-MS/MS for characterization. Based upon the HPLC-MSn results of the digested somatropin, the chelating molecule is mainly bound to a specific lysine amino acid that is located away from the receptor binding site. Therefore, the cell-binding functionality of the characterized NOTA-somatropin is measured, using a HepG2 cell line