Bioanalytical method development and validation of highly selective and sensitive LC-MS/MS method for determination of teriparatide (parathyroid hormone fragment 1-34) in human serum through direct detection of intact teriparatide molecule.

Teriparatide is a novel recombinant peptide fragment of the first 1-34 amino acids of human parathyroid recommended for treatment of osteoporosis. Therapeutic proteins and peptides are routinely estimated using ligand binding assay formats however LC-MS/MS technique which is routinely used in bioanalysis of small molecules has now gained importance in large molecule bioanalysis for the advantages it can offer over LBAs in terms of improved accuracy, selectivity and anti-body free method development. This paper presents a sensitive bioanalytical method for determination of teriparatide in human serum using ultra performance liquid chromatography aligned with tandem mass spectrometric detection. Teriparatide was isolated from human serum using solid phase extraction. The intact peptide was separated on a chromatograph and the multiply charged ion (+7) was detected using a mass spectrometer. The total run time was 4.0 min. The internal standard used was rat PTH 1-34 fragment. The mass transitions of m/z 589.3 > 656.3 for teriparatide and m/z 677.4 > 778.6 for internal standard were used for MS/MS detection. The sample extraction involved a solid phase extraction method followed by concentration of the eluent by evaporation and subsequent reconstitution. The non-specific binding effect caused by the adherence of the peptides/proteins to the vials/tube walls was significantly reduced by using BSA solution as blocking agent. The method has been validated over a linear range of 15.07-913.3 pg/mL with a correlation coefficient ≥ 0.99. The precision (%RSD) was 6.36 to 10.85 and accuracy was within 96.71% to 100.88%. A two-treatment, two-period, cross over study was conducted to establish bioequivalence between test and reference formulation (20 mcg/80 mL - solution for injection) and the method was successfully applied to quantify teriparatide in serum samples of this clinical study and about 1220 human serum samples were analyzed to determine teriparatide. This method is a promising anti-body free LC-MS/MS based methodology for estimation of teriparatide in human serum and may be applied as starting method for other such peptide molecules.

NMR Spectroscopy for Protein Higher Order Structure Similarity Assessment in Formulated Drug Products.

Peptide and protein drug molecules fold into higher order structures (HOS) in formulation and these folded structures are often critical for drug efficacy and safety. Generic or biosimilar drug products (DPs) need to show similar HOS to the reference product. The solution NMR spectroscopy is a non-invasive, chemically and structurally specific analytical method that is ideal for characterizing protein therapeutics in formulation. However, only limited NMR studies have been performed directly on marketed DPs and questions remain on how to quantitively define similarity. Here, NMR spectra were collected on marketed peptide and protein DPs, including calcitonin-salmon, liraglutide, teriparatide, exenatide, insulin glargine and rituximab. The 1D 1H spectral pattern readily revealed protein HOS heterogeneity, exchange and oligomerization in the different formulations. Principal component analysis (PCA) applied to two rituximab DPs showed consistent results with the previously demonstrated similarity metrics of Mahalanobis distance (DM) of 3.3. The 2D 1H-13C HSQC spectral comparison of insulin glargine DPs provided similarity metrics for chemical shift difference (Δδ) and methyl peak profile, i.e., 4 ppb for 1H, 15 ppb for 13C and 98% peaks with equivalent peak height. Finally, 2D 1H-15N sofast HMQC was demonstrated as a sensitive method for comparison of small protein HOS. The application of NMR procedures and chemometric analysis on therapeutic proteins offer quantitative similarity assessments of DPs with practically achievable similarity metrics.

Oral Delivery of Teriparatide Using a Nanoemulsion System: Design, in Vitro and in Vivo Evaluation.

PURPOSE: Investigate the possibility of delivering teriparatide orally using nanoemulsion. METHOD: Teriparatide was allowed to interact with chitosan in the presence of HPßCD.The formed polyelectrolyte complex (PEC) was characterized by DSC, FTIR, DLS and for entrapment efficiency. PEC was the incorporated in an oil phase consisting of Oleic Acid, Labrasol and Plurol Oleique to form a nanoemulsion. This preparation was characterized for refractive index, viscosity, pH, conductivity, particle size, and morphology.Bioavailability of the preparation was evaluated using rabbits against SC injection. The efficacy of the formula was tested using ovariectomized rats (an osteoporosis animal model) and mechanical and histological tests were conducted on their bones. The stability of the preparation was evaluated by storing samples at 4o C, 25o C and 40o C for three months. RESULTS: PEC testing demonstrate a complex formation with particle size of 208 nm, zeta potential of +17 mV and entrapment efficiency of 49%. For the nanoemulsion, the results demonstrate the formation of a nano-sized dispersed system (108 nm) with a drug loading of 98% and a percent protection of 90% and 71% in SGF and SIF respectively. Bioavailability results showed a sustained release profile was achieved following the oral formulation administration. Efficacy studies showed improvement in the strength, thickness and connectivity of bones. Short-term stability study demostrated that the nanoemulsion is mostly stable at 4o C. CONCLUSION: These findings demonstrate the ability of delivering Teriparatide orally using oleic acid based dispersion in combination with chitosan PEC.

Comparative Physicochemical and Biological Characterisation of the Similar Biological Medicinal Product Teriparatide and Its Reference Medicinal Product.

BACKGROUND: In January 2017, the European Commission approved Terrosa® (company code RGB-10) as one of the first biosimilar medicinal products of teriparatide for the same indications as for the reference medicinal product Forsteo® (Lilly France S.A.S.), which has been on the market in the European Union since 2003. The active pharmaceutical ingredient of the reference medicinal product is the biologically active 1-34 fragment of the endogenous human parathyroid hormone [PTH(1-34)]. It is one of the three bone anabolic agents used in the treatment of osteoporosis promoting bone formation and preventing fragility fractures. OBJECTIVE: The objective of this paper is to summarise the results of the comparative analysis of representative batches of both the RGB-10 drug product and the reference medicinal product performed by physicochemical and in vitro biological methods. METHODS: A series of state-of-the-art analytical methods were applied in a comparative head-to-head manner for testing the similarity in respect to purity, content, structure and potency. RESULTS: Based on the results of the comprehensive physicochemical and biological characterisation, RGB-10 proved to be highly similar to the reference medicinal product with respect to the critical quality attributes investigated. CONCLUSION: The results of the quality comparability study demonstrated similarity of RGB-10 to the reference medicinal product, providing the scientific basis for conducting a specifically designed clinical programme, and supported registration of the Marketing Authorisation Application of RGB-10 in the EU.

Hyaluronic Acid Modified Risedronate and Teriparatide Co-loaded Nanocarriers for Improved Osteogenic Differentiation of Osteoblasts for the Treatment of Osteoporosis.

BACKGROUND: Owing to its multifactorial intricate pathogenesis, combined therapeutic regimen is considered appropriate for the treatment of osteoporosis. However, a multi-drug regimen is also associated with adverse effects due to the non-specific distribution of drugs. Therefore, the present study aims for efficient codelivery of risedronate (RDN) (a potent bone anti-resorptive drug) and teriparatide (TPD) (anabolic agent) as hyaluronic acid (HA)-modified chitosan nanoparticles (NPs). METHODS: RDN/TPD NPs were synthesized using the high- pressure homogenization - solvent evaporation technique. The fabricated NPs were then characterized and optimized for suitable physicochemical characteristics. The optimized NPs were then evaluated for bone remodeling potential via assessment of time-mannered modulation in proliferation, differentiation, and mineralization of osteoblasts. RESULTS: Results showed that HA-RDN/TPD NPs exhibited excellent physicochemical characteristics (nanoscopic size, stable zeta potential, high entrapment efficiency, and smooth spherical shape) and remained stable upon storage in the refrigerator. Assessment of various aspects of the cell growth cycle (i.e., proliferation, differentiation, and mineralization) evidenced promising bone regeneration efficacy of HA-RDN/TPD NPs. CONCLUSION: This new strategy of employing simultaneous delivery of anti-resorptive and bone-forming agents would open new horizons for scientists, researchers, and healthcare providers as an efficient pharmacotherapy for the treatment of osteoporosis.

Specific solubilization of impurities in culture media: Arg solution improves purification of pH-responsive tag CspB50 with Teriparatide.

Protein purification using non-chromatographic methods is a simple technique that avoids costly resin. Recently, a cell surface protein B (CspB) tag has been developed for a pH-responsive tag for protein purification by solid-liquid separation. Proteins fused with the CspB tag show reversible insolubilization at acidic pH that can be used in solid-liquid separation for protein purification. However, brown-color impurities from co-precipitation hamper further analysis of the target proteins. In this study, we investigated the effect of additives on the co-precipitation of CspB-tagged Teriparatide (CspB50TEV-Teriparatide) expressed in Corynebacterium glutamicum and associated impurities. Arginine (Arg) at 1.0 M was found to be the most effective additive for removing impurities, particularly carotenoids and nucleic acids. Furthermore, all impurities detected in the fluorescence and absorbance spectra were successfully removed by the repetition of precipitation-redissolution in the Arg solution. The precipitation yield of the CspB50TEV-Teriparatide did not change with the addition of Arg and the repetition of the precipitation-redissolution process. Collectively, our findings indicate that the specific desorption of π-electron rich compounds by Arg may be useful in conjunction with the pH-responsive CspB tag for solid-liquid protein purification.

Inhibiting Sterilization-Induced Oxidation of Large Molecule Therapeutics Packaged in Plastic Parenteral Vials.

For many years, glass has been the default material for parenteral packaging, but the development of advanced plastics such as cyclic olefin polymers and the rapidly increasing importance of biologic drugs have provided new choices, as well as more stringent performance requirements. In particular, many biologics must be stored at non-neutral pH, where glass is susceptible to hydrolysis, metal extraction, and delamination. Plastic containers are not susceptible to these problems, but suffer from higher gas permeability and a propensity for sterilization-induced radical generation, heightening the risk of oxidative damage to sensitive drugs. This study evaluates the properties of a hybrid material, SiOPlas™, in which an ultrathin multilayer coating is applied to the interior of cyclic olefin polymer containers via plasma-enhanced chemical vapor deposition. Our results show that the coating decreases oxygen permeation through the vial walls 33-fold compared to uncoated cyclic olefin polymers, which should allow for improved control of oxygen levels in sensitive formulations. We also measured degradation of two biologic drugs that are known to be sensitive to oxidation, teriparatide and erythropoietin, in gamma and electron beam sterilized SiOPlas™, glass, and uncoated cyclic olefin polymer vials. In both cases, solutions stored in SiOPlas™ vials did not show elevated susceptibility to oxidation compared to either glass or unsterilized controls. Taken together, these results suggest that hybrid materials such as SiOPlas™ are attractive choices for storing high-value biologic drugs.LAY ABSTRACT: One of the most important functions of parenteral drug containers is safeguarding their contents from damage, either chemical or physical. Glass has been the container material of choice for many years, but concerns over breakage and vulnerability to chemical attack at non-neutral pH have spurred the rise of advanced plastics as alternatives. Plastics solve many problems associated with glass but introduce several of their own, including increased gas permeation and generation of oxidizing radicals during sterilization. In this article, we evaluate SiOPlas™, a hybrid material consisting of plastic with a thin multilayer coating applied to the interior, for its ability to overcome these two problems. We find that SiOPlas™ is much less permeable to oxygen than uncoated plastic, and that two biologic drugs stored in gamma and electron beam sterilized SiOPlas™ vials do not display elevated levels of oxidation compared to either glass or unsterilized vials. This suggests that hybrid materials such as SiOPlas™ can exhibit the best qualities of both glass and plastic, making them attractive materials for storing high-value parenteral drugs.

In vitro and preclinical assessment of an intranasal spray formulation of parathyroid hormone PTH 1-34 for the treatment of osteoporosis.

Osteoporosis treatment with PTH 1-34 injections significantly reduces the incidence of bone fracture. Potential further reductions in fracture rate should be observed through nasal spray delivery to address the poor compliance associated with patient dislike of repeated PTH 1-34 subcutaneous injections. In vitro human osteoblast-like Saos-2 cell intracellular cAMP levels were used to define PTH 1-34 nasal spray formulation bioactivity. The chemically synthesised PTH 1-34 had an EC50 of 0.76nM. Absorption enhancers polyethylene glycol (15)-hydroxystearate (Solutol® HS15), poloxamer 407, chitosan or sodium hyaluronate did not diminish the bioactivity of PTH 1-34 within an in vitro cell culture model (p >0.05). We also demonstrated the effectiveness of the transmucosal absorption enhancer Solutol® HS15 in a nasal spray formulation using a preclinical pharmacokinetic model. In Sprague-Dawley rats without the absorption enhancer the uptake of PTH 1-34 into the blood via intranasal delivery produced a Cmax of 2.1±0.5ng/ml compared to 13.7±1.6ng/ml with Solutol® HS15 enhancer (p=0.016) and a Cmax14.8±8ng/ml in subcutaneous injections. Together these data illustrate that the nasal spray formulation bioactivity in vitro is not affected by the nasal spray absorption enhancers investigated, and the Solutol® HS15 nasal spray formulation had an equivalent pharmacokinetic profile to subcutaneous injection in the rat model. The Solutol® HS15 formulation therefore demonstrated potential as a PTH 1-34 nasal spray formulation for the treatment of osteoporosis.

Preparation, characterization and in vivo evaluation of a combination delivery system based on hyaluronic acid/jeffamine hydrogel loaded with PHBV/PLGA blend nanoparticles for prolonged delivery of Teriparatide.

In the current study, biodegradable PHBV/PLGA blend nanoparticles (NPs) containing Teriparatide were loaded in hyaluronic acid/jeffamine (HA-JEF ED-600) hydrogel to prepare a combination delivery system (CDS) for prolonged delivery of Teriparatide. The principal purpose of the present study was to formulate an effective and prolonged Teriparatide delivery system in order to reduce the frequency of injection and thus enhance patient's compliance. Morphological properties, swelling behaviour, crosslinking efficiency and rheological characterization of HA-JEF ED-600 hydrogel were evaluated. The CDS was acquired by adding PHBV/PLGA NPs to HA-JEF ED-600 hydrogel simultaneously with crosslinking reaction. The percentage of NPs incorporation within the hydrogel as well as the loading capacity and morphology of Teriparatide loaded CDS were examined. Intrinsic fluorescence and circular dichroism spectroscopy proved that Teriparatide remains stable after processing. The release profile represented 63% Teriparatide release from CDS within 50days with lower burst release compared to NPs and hydrogel. MTT assay was conducted by using NIH3T3 cell line and no sign of reduction in cell viability was observed. Based on Miller and Tainter method, LD50 of Teriparatide loaded CDS was 131.8mg/kg. In vivo studies demonstrated that Teriparatide loaded CDS could effectively increase serum calcium level after subcutaneous injection in mice. Favourable results in the current study introduced CDS as a promising candidate for controlled delivery of Teriparatide and pave the way for future investigations in the field of designing prolonged delivery systems for other peptides and proteins.

Injectable PLGA/Hydroxyapatite/Chitosan Microcapsules Produced by Supercritical Emulsion Extraction Technology: An In Vitro Study on Teriparatide/Gentamicin Controlled Release.

Supercritical emulsion extraction (SEE) is proposed as a green and effective strategy for the fabrication of chitosan-covered poly-lactic-co-glycolic acid (chi-PLGA) injectable microcapsules for the controlled release of teriparatide (THA) and teriparatide/gentamicin sulfate (THA/Gen). These formulations can be used for locally bone pathologies treatment or in complex fracture healing of aged patients. Several oil-water (o-w) and water-oil-water (w-o-w) emulsions were processed by SEE to produce multifunctional microcapsules containing hydroxyapatite (HA) within a poly-lactic-co-glycolic acid (PLGA) matrix (up to 24 mg/g) and with both THA (0.45 mg/g) and Gen (up to 9 mg/g). Chitosan coating was also successfully added, as external layer (0.4 µm). SEE-fabricated microcapsules showed good encapsulation efficiency (up to 90%) for all the drugs tested and a mean size ranging between 1.4 (±0.4) µm and 2.2 (±0.5) µm. Different drug amounts loaded and microcapsules compositions assured a controlled drug release over a wide range of times and concentrations, as in vitro monitored in PBS medium at 37°C for 15/20 days. HA embedded into the biopolymer structure delayed the THA release profile; chitosan coating strongly reduced the initial drug "burst" release. In addition, the coencapsulation of both THA and Gen, which have very different water solubility, accelerated the release profile of the less water-soluble drug. No drugs degradation was also monitored after the SEE manufacturing. Apparent drug diffusivities (D) were calculated by fitting of the release profiles. In the case of Gen, D ranged between 2.9 × 10(-8) and 1.6 × 10(-9) cm(2)s(-1) if the drug was entrapped in simple PLGA or in the chitosan-coated microcapsules, respectively. In the case of THA, the calculated values ranged between 8.1 × 10(-9) and 7.4 × 10(-10) cm(2)s(-1) when the drug was entrapped in PLGA/HA microcapsules or in the chitosan-coated ones, respectively. These mass transfer values are consistent with the different release behaviors observed and confirmed the possibility of multicomponent microcapsules fabrication by SEE.

Stability of lyophilized teriparatide, PTH(1-34), after reconstitution.

The peptide teriparatide, also known as parathyroid hormone (1-34), PTH(1-34), was developed for intranasal delivery, requiring extended stability of the reconstituted product for up to four weeks at room temperature. Lyophilized formulations of PTH(1-34), containing glycine and trehalose and using lactate as the buffer, are stable for months upon storage. However, the physical stability of the peptide after reconstitution unexpectedly varied considerably, depending on peptide concentration and storage temperature, with precipitation seen within two to four weeks in some samples. By comparison, equivalent samples that did not undergo lyophilization did not display any precipitation upon storage in the liquid state for as long as twelve weeks. PTH(1-34) appears to adopt a higher order structure that is perturbed by the combined stresses of freezing and drying, leading to greater propensity to aggregate, which is accentuated at higher peptide concentrations and at higher temperatures. The precipitation seems to be correlated with increased amounts of subvisible particles. This study shows the importance of peptide conformation in long-term stability and illustrates the ability of lyophilization to cause increased propensity to aggregate, even in a peptide.

Effect of Bone Mineral Density on Rotator Cuff Tear: An Osteoporotic Rabbit Model.

INTRODUCTION: An increased bone mineral density (BMD) in the proximity to tendon insertion can improve rotator cuff repair and healing. However, how a decrease of BMD in the humeral head affects the biomechanical properties of the rotator cuff tendon is still unclear. Previous studies have demonstrated ovariectomy in animals to lead to osteoporosis and decreased BMD, and Teriparatide (PTH) administration to improve BMD and strength of bone. This study aimed to explore the correlation between humeral head BMD and infraspinatus (ISP) tendon insertion strength, and if an increase in bone quantity of the humeral head can improve the strength of the rotator cuff. MATERIALS AND METHODS: Eighteen New England white rabbits were divided into the 3 groups: Control, Ovariectomy-Saline (OVX-Saline), and Ovariectomy-PTH (OVX-PTH). The OVX-Saline group and the OVX-PTH were administered daily saline and Teriparatide injections for 8 weeks starting at 17 weeks of OVX. BMD of the humeral head was measured, the ISP tendon failure load was tested and the failure stress was calculated. One specimen from each group was used for histological analysis. Linear regression analysis was used to derive equations for the BMD and failure stress. RESULTS: Significant differences were observed in the measured humeral head BMD of the Control and OVX-PTH groups compared to the OVX-Saline group (P = 0.0004 and P = 0.0024, respectively). No significant difference was found in failure stress among the three groups, but an expected trend with the control group and OVX-PTH group presenting higher failure strength compared to the OVX-Saline group. BMD at the humeral head showed a positive linear correlation with stress (r2 = 0.54). Histology results showed the superiority in OVX-PTH group ISP enthesis compared to the OVX-Saline group. CONCLUSION: Bone loss of the humeral head leads to decreased tendon/bone insertion strength of the infraspinatus tendon enthesis. Teriparatide administration can increase bone density of the humeral head and may improve the mechanical properties of the infraspinatus tendon enthesis.

Validation of an ultrasensitive LC-MS/MS method for PTH 1-34 in porcine plasma to support a solid dose PK study.

BACKGROUND: The bioanalysis of Teriparatide (PTH 1-34) is extremely challenging due to the low plasma concentrations present at a therapeutic level. An LC-MS/MS-based method was developed that detected PTH 1-34 at 15 pg/ml in porcine plasma, and was validated using the bioanalytical method validation guidelines. RESULTS: The analytical methodology demonstrated good linearity over a range of 15-1000 pg/ml, and demonstrated good precision and accuracy. The validated method was used to support a trial comparing a solid state dose to a solution-based injection (Forteo™). CONCLUSION: The ability to quantify the peptide at low pg/ml in porcine plasma demonstrates that it is possible to develop very sensitive LC-MS/MS-based methodologies to support the bioanalysis of large peptide biotherapeutics.

Reversible Deterioration in Hypophosphatasia Caused by Renal Failure With Bisphosphonate Treatment.

Hypophosphatasia is an inborn error of metabolism caused by mutations in the ALPL gene. It is characterized by low serum alkaline phosphatase (ALP) activity and defective mineralization of bone, but the phenotype varies greatly in severity depending on the degree of residual enzyme activity. We describe a man with compound heterozygous mutations in ALPL, but no previous bone disease, who suffered numerous disabling fractures after he developed progressive renal failure (for which he eventually needed dialysis treatment) and was prescribed alendronate treatment. A bone biopsy showed marked osteomalacia with low osteoblast numbers and greatly elevated pyrophosphate concentrations at mineralizing surfaces. In vitro testing showed that one mutation, T117H, produced an ALP protein with almost no enzyme activity; the second, G438S, produced a protein with normal activity, but its activity was inhibited by raising the media phosphate concentration, suggesting that phosphate retention (attributable to uremia) could have contributed to the phenotypic change, although a pathogenic effect of bisphosphonate treatment is also likely. Alendronate treatment was discontinued and, while a suitable kidney donor was sought, the patient was treated for 6 months with teriparatide, which significantly reduced the osteomalacia. Eighteen months after successful renal transplantation, the patient was free of symptoms and the scintigraphic bone lesions had resolved. A third bone biopsy showed marked hyperosteoidosis but with plentiful new bone formation and a normal bone formation rate. This case illustrates how pharmacological (bisphosphonate treatment) and physiologic (renal failure) changes in the "environment" can dramatically affect the phenotype of a genetic disorder.

PEGylation site-dependent structural heterogeneity study of monoPEGylated human parathyroid hormone fragment hPTH(1-34).

The structures of C- and N-terminally monoPEGylated human parathyroid hormone fragment hPTH(1-34) as well as their unmodified counterparts, poly(ethylene glycol) (PEG) and hPTH(1-34), have been studied by small-angle neutron scattering (SANS). The scattering results show that free hPTH(1-34) in 100 mM phosphate buffer (pH 7.4) aggregates into clusters. After conjugation with PEG, the PEG-peptide conjugates self-assemble into a supramolecular core-shell structure with a cylindrical shape. The PEG chains form a shell around the hPTH(1-34) core to shield hPTH(1-34) from the solvent. The detailed structural information on the self-assembled structures is extracted from SANS using a model of the cylindrical core with a shell of Gaussian chains attached to the core surface. On the basis of the data, because of the charge-dipole interactions between the conjugated PEG chain and the peptide, the conjugated PEG chain forms a more collapsed conformation compared to free PEG. Moreover, the size of the self-assembled structures formed by the C-terminally monoPEGylated hPTH(1-34) is about 3 times larger than that of the N-terminally monoPEGylated hPTH(1-34). The different aggregation numbers of the self-assembled structures, triggered by different PEGylation sites, are reported. These size discrepancies because of different PEGylation sites could potentially affect the pharmacokinetics of the hPTH(1-34) drug.

Intrinsic bioconjugation for site-specific protein PEGylation at N-terminal serine.

Recently developed chemical ligation protocols were elaborated for rapid N-terminal protein PEGylation. We introduce a PEG-salicylaldehyde ester and demonstrate its site-specific ligation to the N-terminus of the RNase S protein and to the therapeutic polypeptide PTH (1-34).

Semisynthesis of peptoid-protein hybrids by chemical ligation at serine.

Chemical ligation protocols were explored for generating semisynthetic peptoid-protein hybrid architectures containing a native serine residue at the ligation site. Peptoid oligomers bearing C-terminal salicylaldehyde esters were synthesized and ligated to the N-terminus of the RNase S protein or the therapeutic hormone PTH(1-34) polypeptide. This technique will expand the repertoire of strategies to enable design of hybrid macromolecules with novel structures and functions not accessible to fully biosynthesized proteins.

One-pot peptide ligation-desulfurization at glutamate.

An efficient methodology for ligation at glutamate (Glu) is described. A γ-thiol-Glu building block was accessed in only three steps from protected glutamic acid and could be incorporated at the N-terminus of peptides. The application of these peptides in one-pot ligation-desulfurization chemistry is demonstrated with a range of peptide thioesters, and the utility of this methodology is highlighted through the synthesis of the osteoporosis peptide drug teriparatide (Forteo).

High sensitivity LC-MS/MS method for direct quantification of human parathyroid 1-34 (teriparatide) in human plasma.

Teriparatide, the 1-34 fragment of human parathyroid hormone, is used to treat osteoporosis patients with a high risk of fracture by stimulating new bone formation. Routinely teriparatide is quantified using radioimmunoassay however the LC-MS/MS described here has the potential to achieve greater accuracy and precision, higher specificity, and is readily implemented in routine bioanalytical laboratories. Hence a complete method combining effective sample prep with appropriate LC separation and selected reaction monitoring (SRM) MS detection was developed to selectively separate teriparatide from closely related endogenous peptides and to reduce interferences. Samples were concentrated without evaporation, minimizing the risk of adsorptive losses. Chromatography was performed on a sub 2µm particle charged surface hybrid column, which provided significantly higher peak capacity than a traditional C18 column when formic acid was used as the mobile phase modifier. Total LC cycle time was 6min. An LOD of 15pg/mL (3.6fmol/mL) from 200µL of human plasma was readily achieved and standard curves were accurate and precise from 15pg/mL to 500pg/mL. Mean QC accuracies ranged from 90% to 106%. Mean QC precision was better than 7%. The CV of matrix factors across 6 sources of human plasma was 5%. The assay presented here is the first LC-MS method which reaches clinically relevant detection limits for teriparatide.

Time-resolved fluorescence ligand binding for G protein-coupled receptors.

G protein-coupled receptors (GPCRs) and their ligands are traditionally characterized by radioligand-binding experiments. These experiments yield excellent quantitative data, but have low temporal and spatial resolution. In addition, the use of radioligands presents safety concerns. Here we provide a general procedure for an alternative approach with high temporal and spatial resolution, based on Tb(+)-labeled fluorescent receptor ligands and time-resolved fluorescence resonance energy transfer (TR-FRET). This protocol and its design are detailed here for the parathyroid hormone receptor, a class B GPCR, and its fluorescently labeled 34-amino acid peptide ligand, but it can be easily modified for other receptors and their appropriately labeled ligands. We discuss three protocol options that use Tb(+)-labeled fluorescent ligands: a time-resolved fluorescence separation option that works on native receptors but requires separation of bound and unbound ligand; a TR-FRET option using SNAP-tag-labeled receptors for high-throughput screening; and a TR-FRET option that uses fluorescently labeled antibodies directed against an epitope engineered into the Flag-labeled receptors' N terminus. These protocol options can be used as standard procedures with very high signal-to-background ratios in order to characterize ligands and their receptors in living cells and in cell membranes via straightforward plate-reader measurements.