Impurities identification and quantification for calcitonin salmon by liquid chromatography-high resolution mass spectrometry.

Calcitonin salmon is an important peptide pharmaceutical, which is mainly used for the treatment of osteoporosis and hypercalcemia. Structurally related peptide impurities in a peptide pharmaceutical probably have side effect or even toxicity, thus needs to be carefully characterized according to pharmacopoeia. With the improvement of analytical techniques, liquid chromatography-high resolution mass spectrometry (LC-HRMS) has become a pivotal technique for the identification and quantification of structurally related peptide impurities in peptide materials. In this study, an LC-HRMS-based method has been developed for the identification and quantification of structurally related peptide impurities in calcitonin salmon material. With this method, 7 peptide impurities (> 1 mg/g) in United States Pharmacopoeia (USP) reference standard and 9 peptide impurities (> 1 mg/g) in European Pharmacopoeia (EP) reference standard were identified and accurately quantified. Besides the peptide impurities reported by USP and EP, several new impurities such as [7-Dehydroalanine] calcitonin salmon, triple-sulfate-calcitonin salmon, [26-Proline] calcitonin salmon, [14-Glutamic acid] calcitonin salmon, [20-Glutamic acid] calcitonin salmon, [26-Aspartic acid] calcitonin salmon, calcitonin salmon acid were observed in the reference standard materials studied. The total mass fractions of all structurally related peptide impurities in calcitonin salmon study materials were estimated to be 57.4 mg/g for USP and 46.3 mg/g for EP with associated expended uncertainties at a 95 % confidence level of 5.2 mg/g (k = 2) and 3.1 mg/g (k = 2), respectively.

Low-picomolar, label-free procalcitonin analytical detection with an electrolyte-gated organic field-effect transistor based electronic immunosensor.

Herein a label-free immunosensor based on electrolyte-gated organic field-effect transistor (EGOFET) was developed for the detection of procalcitonin (PCT), a sepsis marker. Antibodies specific to PCT were immobilized on the poly-3-hexylthiophene (P3HT) organic semiconductor surface through direct physical adsorption followed by a post-treatment with bovine serum albumin (BSA) which served as the blocking agent to prevent non-specific adsorption. Antibodies together with BSA (forming the whole biorecognition layer) served to selectively capture the procalcitonin target analyte. The entire immunosensor fabrication process was fast, requiring overall 45min to be completed before analyte sensing. The EGOFET immunosensor showed excellent electrical properties, comparable to those of bare P3HT based EGOFET confirming reliable biosensing with bio-functional EGOFET immunosensor. The detection limit of the immunosensor was as low as 2.2pM and within a range of clinical relevance. The relative standard deviation of the individual calibration data points, measured on immunosensors fabricated on different chips (reproducibility error) was below 7%. The developed immunosensor showed high selectivity to the PCT analyte which was evident through control experiments. This report of PCT detection is first of its kind among the electronic sensors based on EGOFETs. The developed sensor is versatile and compatible with low-cost fabrication techniques.

Biochemical-immunological hybrid biosensor based on two-dimensional chromatography for on-site sepsis diagnosis.

A hybrid-biosensor system that can simultaneously fulfill the immunoassay for protein markers (e.g., C-reactive protein (CRP) and procalcitonin (PCT)) and the enzyme assay for metabolic substances (e.g., lactate) in the same sepsis-based sample has been devised. Such a challenge was pursued through the installation of an enzyme-reaction zone on the signal pad of the typical immuno-strip for the rapid two-dimensional (2-D)-chromatography test. To minimize the mutual interference in the hybrid assays, a pre-determined membrane site was etched in a pattern and mounted with a biochemical-reaction pad, thereby allowing a loaded sample to enter and then stay in the pad for a colored-signal production over the course of an immunoassay. By employing such a constructed system, a serum sample was analyzed according to the vertical direction flowing along the strip, which supplied lactate to the biochemical-reaction zone and then protein markers to the immunological-binding area that was pre-coated with capture antibodies. Thereafter, the enzyme-signal tracers for the immunoassay and the substrate solution were sequentially furnished using a horizontal path for the tracing of the immune complexes that were formed with CRP or PCT. The color signal that was produced from each assay was detected at a pre-determined time and quantified on a smartphone-based detector. Under the optimal conditions, the dynamic ranges for the analytes covered the respective clinical ranges, and the total coefficient of variation was between 8.6% and 13.3%. The hybrid biosensor further showed a high correlation (R2 > 0.95) with the reference systems for the target markers.

Effective Strategies for Diagnosis of Systemic Inflammatory Response Syndrome (SIRS) due to Bacterial Infection in Surgical Patients.

Surgery associated with trauma and soft tissue injuries after surgery significantly activates the systemic immune response. If an infection after surgery occurs, the response is even stronger. Due to spontaneous activation of immune response and elevated biomarkers for sepsis and cytokines, posttraumatic complications such as new-coming postoperative infections are difficult to diagnose. Sepsis as systemic inflammatory response syndrome (SIRS) rapidly progresses through severe sepsis to septic shock and organ failure, and with no applied antibiotic treatment, the disease often ends at death of the patients. In the treatment of non-surgery patients, the biomarkers like white cell blood count, C-reactive protein (CRP) or procalcitonin (PCT) proved to be useful in sepsis recognition. However, diagnostics after surgeries are more complicated and these biomarkers are not ideal. The solution is a sepsis biomarker, which would have high sensitivity and specificity, that can improve diagnostic accuracy of sepsis, should also be measured easily by the patients, and should not be too expensive. We think more sensitive and specific biomarkers such as presepsin (sCD14-ST) or CD64 index on neutrophils could be useful. A diagnosis of sepsis should be based on clinical signs, and clinicians should use biomarker that is not only most sensitive and specific but also is cost effective. Furthermore, confirmation of the bacterial or fungal infection with blood cultures or with the use of broad range polymerase chain reaction (PCR), when culturing is impossible, should be performed.

Lung cancer may increase serum procalcitonin level.

INTRODUCTION: Serum procalcitonin (PCT) is a biomarker used routinely to diagnose infections. Some malignancies are usual false positives for PCT. However, its value and behavior in the setting of lung cancers are poorly known. The objective of this study was to assess PCT positivity in a lung cancer cases series. METHOD: Between November 2011 and September 2012, all cases of newly diagnosed lung cancer with a pre-antineoplastic PCT assay and no patent signs of infection were included in the study. All PCT levels were assessed by immunofluorescent assay in a single laboratory. RESULTS: Eighty-nine patients were included (70.8% male; mean age 62; small-cell cancer 20.2%; stage IV cancer 60.7%). Overall, PCT was positive in 42%. A neuroendocrine component, having 2 or more metastatic sites, having a pleura or a liver metastasis, and being positive for CRP were all significantly associated with positive PCT in univariate analysis. In multivariate analysis, only the presence of a neuroendocrine component remained strongly associated with a positive PCT (AOR=7.24 [CI=95% 1.91-27.51]; P=0.004). Finally, baseline PCT levels <0.5 µg/l were found in 43% of NSCLC with a neuroendocrine component, vs. 9% of cancers with other histology (P=0.0001). CONCLUSION: Lung cancer may cause false positives for procalcitonin, particularly in cases of neuroendocrine cancers or in the presence of multiple metastases. These results should be taken into account for PCT-based decisional algorithms.

[The value of quantitative analysis of procalcitonine in diagnostics of septic complications in patients with autoimmune rheumatic diseases].

The infections very often complicate the course of autoimmune rheumatic diseases. In diagnostic of septic complications in rheumatic patients the new biomarkers of infections can have a decisive importance. The procalciotonine test is one of them. The issue was to evaluate the diagnostic informativity of this test. The sample included 93 patients. The examination was applied to 65 patients with rheumatic diseases. Among them, 13 patients had bacterial infections. The group consisted of 33 patients with rheumatoid arthritis, 11 patients with systemic lupus erythematous, 6 patients with systemic angiitis, and 15 patients with other rheumatic diseases. The comparative group included 27 patients of cardio-therapeutic profile and 8 of these patients had bacterial infections. The procalcitonine test was applied with quantitative electrochemiluminescent technique. In patients with rheumatoid arthritis the mean levels of procalciotonine test consisted 0.10 +/- 0.13 ng/ml; with systemic lupus erythematous--0.08 +/- 0.06 ng/ml; with systemic angiitis--0.22 +/- 0.2 ng/ml; with other rheumatic diseases--0.12 +/- 0.15 ng/ml; of cardio-therapeutic profile without infections--0.08 +/- 0.06 ng/vl/ With threshold of procalcitonine test higher than 0.5/ml the sensitivity to diagnostic of infections consisted of 58%, specificity--94% in the group with rheumatic diseases. The procalciotonine test in case of no infection process with values higher than 0.5 ng/ml was detected in three patients. The evaluation of dependence of sensitivity and specificity for procalciotonine test and C-reactive protein the area under curve of procalcitonine test was larger in patients with rheumatic diseases (0.85 against 0.79) and in patients of cardio-therapeutic profile (0.92 against 0.90). The quantitative procalcitonine test is the best technique to detect septic complications in rheumatic patients.

Targeting procalcitonin with novel murine monoclonal antibodies.

The aim of this study was to produce monoclonal and polyclonal antibodies against procalcitonin (PCT), a valid post-mortem marker of sepsis marker. Monoclonal antibodies (MAbs) were made from hyperimmune Balb/c mice by injecting 50 microg of purified antigen. These mice were immunized four times and given a final boost, and their spleen cells were collected and fused with SP2/0 myeloma cells under the presence of PEG 1450. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using purified protein. Lastly, five MAbs were obtained and designated successfully and were characterized by ELISA and Western immunoblotting. By Western immunoblotting, MAbs were found reactive with the patient's serum specifically. The results showed that the monoclonal antibodies could be used for various pathophysiological analyses in further investigations of procalcitonin and in preparing diagnostic kits.

Comparison of reversible and nonreversible aqueous-soluble lipidized conjugates of salmon calcitonin.

Reversible aqueous lipidization (REAL) at the interdisulfide bond has been shown to improve the deliverability of some peptide drugs. Recently, we developed a nonreversible aqueous lipidization method targeted at the interdisulfide bond of salmon calcitonin (sCT). The resultant derivative had comparable hypocalcemic activity to sCT after subcutaneous injection in rats, despite possessing significantly different biophysical properties. The purpose of this study was to conduct a comparative evaluation of the biophysical properties of the reversible aqueous-soluble lipidized sCT (REAL-sCT) and its corresponding nonreversible aqueous-soluble compound (Mal-sCT) with a view to correlate these properties to the bioactivities of the peptides. REAL-sCT and Mal-sCT were successfully synthesized, purified and identified. Both conjugates showed comparable retention times in a C-18 HPLC column, as well as robust helical structures and aggregation behavior in water, although REAL-sCT was shown by dynamic light scattering experiments to form larger aggregates than Mal-sCT in water. The larger particle size of REAL-sCT correlated with its stronger resistance to degradation by intestinal enzymes. Unlike Mal-sCT, REAL-sCT was rapidly converted to sCT in liver juice; however, the regenerated sCT appeared to degrade at a slower rate than unmodified sCT in the liver juice. Compared with sCT, REAL-sCT after subcutaneous injection as an aqueous solution at a dose of 0.15 mg/kg produced a prolonged hypocalcemic activity that lasted at least 24 h in the rat. Using a novel LC-MS/MS method that was developed for this study, we were able to show concomitant increases in REAL-sCT and sCT plasma concentrations with time, the latter prevailing at 10% the molar concentration of the former. In contrast, sCT was not present in the plasma following the subcutaneous injection of Mal-sCT, although a comparable hypocalcemic activity with shorter duration was observed. Oral administration of REAL-sCT and Mal-sCT as aqueous solutions at sCT equivalent dose of 5.0 mg/kg did not produce significant hypocalcemic activity. This study is the first thorough examination of the biophysical characteristics of the corresponding reversible and nonreversible aqueous-soluble lipidized peptide molecules. The results obtained should be useful for the development of the oral formulation of peptide and protein drugs.

Improvement in industrial re-chromatography (recycling) procedure in solvent gradient bio-separation processes.

Re-chromatography or recycling impure products obtained from the batch runs of solvent gradient chromatography is commonly practiced in industry to improve product yield. However, as the re-chromatography steps are carried out at the expense of running fresh batches, any improvement in the yield comes as a trade-off with the production time, and hence productivity. In recent studies, on the other hand, it has been suggested that with a properly designed recycling process one can not only improve the yield, but the productivity as well. That study, however, considered a steady-state recycling process, a technology yet to be implemented with bio-chromatographic systems. In the present paper we are reporting a study made on non-steady-state recycling or re-chromatography, as it is typically done in industrial practice. The results point out an amendment to the standard way of designing solvent gradients, which is necessary to improve both the yield and the productivity of an industrial run with recycle. Although the test case used here was the separation of an industrial peptide, Calcitonin, in a reversed-phase column, the general methodology of gradient manipulation, needless to say, is also valid for other solvent gradient processes like ion-exchange, HIC, etc.

Role of recycling in improving the performance of chromatographic solvent gradient purifications.

With significant advancement in the upstream processing technology, downstream processing of large bio-molecules is becoming the bottle-neck in the production chain. To face this challenge, design and development of efficient separation processes has become crucial. As a step towards boosting the performance of a chromatographic separation process through improved design, we investigated the potential of recycling as a process option. The most important advantage of recycling is that it can be implemented in an existing batch system without any major investment and consultation. Although impure products are recycled in industries, it is done as additional batch, and only then, when the recoverable product is valuable enough to surpass the loss of productivity in running the additional batches. In our study, on the other hand, it was found that a well-designed recycle can not only improve the yield, but also the productivity of a multi-component purification. A series of multiobjective optimization studies were carried out on multi-component separation to comprehend the role of recycling with reference to an industrially relevant problem, i.e. the chromatographic purification step of the production process of calcitonin.

Amino acid sequence and biological activity of a calcitonin-like diuretic hormone (DH31) from Rhodnius prolixus.

Diuresis in the blood-gorging hemipteran Rhodnius prolixus is under neurohormonal control and involves a variety of processes and tissues. These include ion and water movement across the epithelium of the crop and the Malpighian tubules, and muscle contractions of the crop, hindgut and dorsal vessel, which facilitate mixing of the blood-meal, mixing of the haemolymph, as well as the expulsion of waste. One of the neurohormones that might play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH(31)) family of insect peptides. Previously we have demonstrated the presence of DH(31)-like peptides in the central nervous system (CNS) and gut of R. prolixus 5th instars. In the present work, a DH(31) from the CNS of 5th instar R. prolixus was isolated using reversed-phase liquid chromatography (RPLC), monitored with an enzyme-linked immunosorbent assay (ELISA) combined with matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, and sequenced using tandem mass spectrometry and Edman degradation. This neuropeptide is the first to be sequenced in R. prolixus and has a sequence identical to that found previously for Dippu-DH(31) from the cockroach Diploptera punctata. In previous studies testing Rhopr/Dippu-DH(31) in Malpighian tubule secretion assays, we demonstrated increases in the rate of secretion that were small, relative to that induced by serotonin, but nevertheless 14-fold over baseline. In the present study, we investigated second messenger pathways in response to Rhopr/Dippu-DH(31) and found no increase or decrease in cyclic adenosine monophosphate (cyclic AMP) content of the Malpighian tubules. DH(31)-like immunoreactivity is present over the dorsal hindgut, anterior dorsal vessel and dorsal diaphragm, and bioassays of the R. prolixus dorsal vessel and hindgut indicate that Rhopr/Dippu-DH(31) increases the frequency of muscle contractions of both tissues. Second messenger pathways were also investigated for the dorsal vessel and hindgut.

Parametric study of a 6-column countercurrent solvent gradient purification (MCSGP) unit.

The novel "multicolumn countercurrent solvent gradient purification" (MCSGP) process has been modeled for the purification of a polypeptide mixture characterized by a strong non-linear competitive adsorption isotherm. As a model system, the purification of an industrial polypeptide mixture containing 46% of the hormone calcitonin has been selected. The many impurities contained in the mixture have been lumped into three key impurities, which are selected as the ones eluting closer to the main component. The simulation model allows for a better understanding of the complex operating behavior of the multicolumn system, which has been experimentally investigated in a previous work. Through a systematic parametric analyses of the model behavior, the main operating parameters controlling the process performance in terms of purity and yield are investigated. The study of internal liquid and adsorbed phase concentration profiles along the unit for the different operating conditions allow elucidating the working principle of the new separation process. It is found that the MCSGP unit achieves much higher yields for a given product purity than the corresponding single-column batch units.

A continuous multicolumn countercurrent solvent gradient purification (MCSGP) process.

Biomolecules are often purified via solvent gradient batch chromatography. Typically suitable smooth linear solvent gradients are applied to obtain the separation between the desired component and hundreds of impurities. The desired product is usually intermediate between weakly and strongly adsorbing impurities, and therefore a central cut is required to get the desired pure product. The stationary phases used for preparative and industrial separations have a low efficiency due to strong axial dispersion and strong mass transfer resistances. Therefore a satisfactory purification often cannot be achieved in a single chromatographic step. For large scale productions and for very valuable molecules, countercurrent operation such as the well known SMB process, is needed in order to increase separation efficiency, yield and productivity. In this work a novel multicolumn solvent gradient purification process (MCSGP-process) is introduced, which combines two chromatographic separation techniques, which are solvent gradient batch and continuous countercurrent SMB. The process consists of several chromatographic columns, which are switched in position opposite to the flow direction. Most of the columns are equipped with a gradient pump to adjust the modifier concentration at the column inlet. Some columns are interconnected, so that non pure product streams are internally, countercurrently recycled. Other columns are short circuited and operate in batch mode. As a working example the purification of an industrial stream containing 46% of the hormone Calcitonin is considered. It is found that for the required purity the MCSGP unit achieves a yield close to 100% compared to a maximum value of a single column batch chromatography of 66%.

Overexpression and purification of recombinant eel calcitonin and its phylogenetic analysis.

Calcitonin (CT), a peptide hormone that is widely used for the treatment of osteoporosis, Paget's disease, hypercalcemic shock and chronic pain in terminal cancer patients, is produced by the para-follicular cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish. Fish calcitonin, like eel calcitonin (eCT), is more potent and longer lasting than human CT and is one of the many bioactive peptides that require C-terminal amidation for full biological activity. In this study we describe the over-expression and over-production of C-terminal amidated eCT in recombinant Streptomyces avermitilis. A phylogenetic analysis was performed with all the known CT amino acid sequences.

Overexpression, purification and characterization of recombinant salmon calcitonin, a therapeutic protein, in Streptomyces avermitilis.

Calcitonin (CT) is a peptide hormone produced by the parafollicular cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish. Salmon calcitonin (sCT), which is more potent and longer lasting than human CT, has been used widely for the treatment of osteoporosis, paget's disease, hypercalcemic shock and chronic pain in terminal cancer patients. sCT is one of the many bioactive peptides that require C-terminal amidation for full biological activity. In this study we describe the over-expression and over-production of C-terminal amidated sCT in recombinant Streptomyces avermitilis. With this approach the utilization of expensive peptide synthesis can be circumvented.

Delimitation of the time of death by immunohistochemical detection of calcitonin.

To improve the possibilities of delimitating the time of death after longer laytime it was examined if this is possible by immunohistochemical detection of calcitonin. The results show that in our examination material the c-cells of the thyroid glands of up to 4-day-old corpses produce a positive immunoreaction towards calcitonin in all cases whereas none of the corpses older than 13 days show such a reaction. This means that in the case of a negative immunoreaction the time of death can be assumed to lie >4 days before the autopsy. The fact that a negative immunoreaction occurred consistently after 13 days leads to the conclusion that when calcitonin has been stained in a specimen, the death of the respective person must lie a maximum of 12 days earlier, whereby these time-limits may change in considerably different surrounding conditions.

Isolation and characterization of serum procalcitonin from patients with sepsis.

Procalcitonin (PCT) is one of the precursors in the synthesis of calcitonin in thyroidal C-cells and other neuroendocrine cells. PCT and other calcitonin precursors are elevated in the serum of many conditions leading to systemic inflammatory response syndrome. The measurement of PCT in patients suffering from severe bacterial infections is a useful tool for the diagnosis of sepsis. Furthermore, therapeutic decisions are often based on the increase or decline of serum PCT levels. PCT was reported to have 116 amino acids. The aim of our study was the determination of the primary structure of serum PCT from septic patients. Sera containing high PCT-concentrations (>100 ng/ml) were collected from 22 patients with severe sepsis and were pooled for further purification (12.7 microg total concentration of PCT). Pooled PCT was purified on a CT 21-immunoaffinity column, further purified by reversed phase HPLC, and the resulting pure PCT was digested with endoproteinase Asp-N. N-terminal Edman sequencing showed that the first two amino acids (Ala-Pro) of the proposed pro-peptide were missing. Further analyses by MALDI-TOF mass spectroscopy resulted in a distinct mass signal of 12640 Da +/- 0.1%, which is in concordance with the theoretical molecular weight of the N-terminal truncated form (12628 Da). As opposed to previous suggestions, we could not detect any chemical modifications of PCT. In summary, we could demonstrate that PCT in the serum of septic patients is a peptide of only 114 amino acids, instead of the predicted 116 amino acids, lacking the N-terminal dipeptide Ala-Pro. This information on the primary structure of PCT might help in further studies on the physiological role of PCT during sepsis.

Synthesis of biologically active tritiated amylin and salmon calcitonin analogues.

Amylin is a hormone belonging to the calcitonin protein family of peptides. To facilitate receptor screening studies, alternatively radiolabeled and biologically active amylin and salmon calcitonin analogues were synthesized by reductive methylation. Free amino groups of amylin and salmon calcitonin were methylated by reaction of peptides with formaldehyde and sodium [(3)H]borohydride. Radioactively labeled peptides were purified by size exclusion chromatography followed by HPLC. Analysis by MALDI-TOF mass spectrometry of purified amylin and salmon calcitonin peptides revealed incorporation of both two and four tritiated methyl groups per peptide molecule. Specific activities of 22.6 and 23.2 GBq/mmol were measured for amylin and salmon calcitonin, respectively. Methylation of rat amylin and salmon calcitonin did not affect their biological activities as both retained their potency to inhibit insulin-stimulated glycogen synthesis in isolated rat soleus muscle. The synthesis of these tritiated analogues provides an alternative chemically stable radiolabeled ligand which may be useful in exploring receptor interactions within the calcitonin peptide family.

Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects.

Insect diuretic hormones are crucial for control of water balance. We isolated from the cockroach Diploptera punctata two diuretic hormones (DH), Dippu-DH(31) and Dippu-DH(46), which increase cAMP production and fluid secretion in Malpighian tubules of several insect species. Dippu-DH(31) and -DH(46) contain 31 and 46 amino acids, respectively. Dippu-DH(46) belongs to the corticotropin-releasing factor (CRF)-like insect DH family, whereas Dippu-DH(31) has little sequence similarity to the CRF-like DH, but is similar to the calcitonin family. Dippu-DH(46) and -DH(31) have synergistic effects in D. punctata but have only additive effects in Locusta migratoria. Dippu-DH(31) represents a distinct type of insect DH with actions that differ from those of previously identified insect peptides with diuretic activity.