An overview of mechanistic modeling of liquid chromatography

Abstract

Liquid chromatography is considered to be the bottleneck for purification of therapeutic proteins. Development and optimization of chromatography process is a cumbersome activity due to the increasing complexities in the types and content of impurities present in the high product titer cell culture harvest obtained from the upstream processing. Further, regulatory expectations are continuously rising with the recent initiatives of quality by design and process analytical technology expecting the manufacturer to have a deeper understanding of the process and the product. Mechanistic modeling is one approach to gain this deeper understanding of a process step. It involves modeling of the underlying physicochemical processes. A well calibrated model with acceptable predictability can be very effective in both process optimization and process characterization activities. In this paper we provide an overview of mechanistic modeling of liquid chromatography. We discuss the various components that such a model entails and also presents the status quo of this area.     

Predation risk and mating behavior: the responses of moths to bat-like ultrasound

In the presence of predators, animals may reduce or alter theirmating activities. There has been little experimental studyof whether mating behavior varies with the level of predationrisk. Two species of moths, Pseudaletia unipuncta (Noctuidae)and Ostrinia nubilalis (Pyralidae), significantly reduced theirmate-seeking behavior under high levels of simulated predationrisk. Male moths aborted upwind flight in a pheromone plume,and females stopped releasing pheromone in response to soundssimulating the echolocation calls of bats. For O. nubilalis,but not for P. unipuncta, the response varied significantlywith the level of predation risk. Interspecific differencesin behavioral responses likely reflect differences in physiologicalauditory sensitivity and/or behavioral thresholds. Female behavioralresponses to sounds simulating the calls of bats that gleantheir prey from surfaces were significantly weaker than theirresponses to sounds resembling the calls of aerially hawkingbats; these results support the neurophysiological data thatthe calls of gleaning bats are relatively inaudible to moths.This study indicates that some animals can modify their reproductiveactivities in response to auditory cues from predators.     

Determination of 3-keto-4-ene steroids and their hydroxylated metabolites catalyzed by recombinant human cytochrome P450 1B1 enzyme using gas chromatography-mass spectrometry with trimethylsilyl derivatization

A protocol utilizing gas chromatography with selected ion monitoring mass spectrometric detection (GC-SIM-MS) using a simplified trimethylsilyl (TMS) derivatization protocol was developed and validated for the determination of hydroxylated metabolites of 3-keto-4-ene steroids such as testosterone, progesterone and androstenedione. Hydroxylated metabolites catalyzed by human CYP1B1 were extracted with methylene chloride and derivatized with BSTFA-10% TMCS. To get an optimal derivatizing condition, the effect of various incubation times and temperatures was evaluated. When the incubation temperature and time in the presence of the TMS derivatizing agent were increased, the 3-keto group became derivatized with TMS to form a 3-TMS derivative. To minimize the formation of the TMS ether on the 3-keto group, a reaction condition of 56 degrees C for 10 min was used for the routine measurement of the steroids and their hydroxylated metabolite. Performance studies including linearity of calibration curves, extraction efficiency and precision were performed. Linearity of the calibration curves was satisfactory from 0.125 to 5 microM for most compounds except 21-hydroxyprogesterone and 16alpha-hydroxyandrostenedione which deviated from linearity at the lower concentrations. Mean percentage extraction recoveries were greater than 80% for all compounds. Most compounds showed good precisions with C.V.s of within-day precision of less than 5% and C.V.s of between-day precision of less than 10%. The selected ion chromatograms from the recombinant human CYP1B1 incubations with testosterone, progesterone and androstenedione showed evidence of 6beta-, 16alpha-, 2alpha-, and 15alpha-hydroxytestosterone, 6alpha- and 16alpha-hydroxyprogesterone and 6alpha- and 16alpha-hydroxyandrostenedione, respectively. There was no significant interference associated with Escherichia coli membrane extracts in detecting hydroxylated metabolites. This procedure provides a rapid and sensitive method for the evaluation of steroid hydroxylation by CYP isoenzymes.     

Plasmonic Properties of Gold Nanostructures on Gold Film

This paper reports on a systematic study of the plasmonic properties of periodic arrays of gold cylindrical nanoparticles in contact with a gold thin film. Depending on the gold film thickness, it observes several plasmon bands. Using a simple analytical model, it is able to assign all these modes and determine that they are due to the coupling of the grating diffraction orders with the propagating surface plasmons travelling along the film. With finite difference time domain (FDTD) simulations, it demonstrates that large field enhancement occurs at the surface of the nanocylinders due to the resonant excitation of these modes. By tilting the sample, it also observes the evolution of the spectral position of these modes and their tuning through nearly the whole visible range is possible. Such plasmonic substrates combining both advantages of the propagative and localised surface plasmons could have large applications in enhanced spectroscopies.

     

Correlation of hepcidin and serum ferritin levels in thalassemia patients at Chiang Mai University Hospital

Hepcidin is a key iron-regulatory hormone, the production of which is controlled by iron stores, inflammation, hypoxia and erythropoiesis. The regulation of iron by hepcidin is of clinical importance in thalassemia patients in which anemia occurs along with iron overload. The present study aimed to evaluate the correlation between serum hepcidin and ferritin levels in thalassemia patients. This cross-sectional study investigated 64 patients with thalassemia; 16 β-thalassemia major (BTM), 31 β-thalassemia/hemoglobin (Hb) E (BE), and 17 Hb H + AE Bart’s disease (Hb H + AE Bart’s). The levels of serum hepcidin and ferritin, and Hb of the three groups were measured. The median values of serum ferritin and Hb were significantly different among the three groups, whereas serum hepcidin values were not observed to be significantly different. The correlation of the serum hepcidin and ferritin levels was not statistically significant in any of the three groups of thalassemia patients with BTM, BE, or Hb H + AE Bart’s (r = −0.141, 0.065 and −0.016, respectively). In conclusion, no statistically significant correlations were observed between serum hepcidin with any variables including serum ferritin, Hb, age, labile plasma iron (LPI), and number of blood transfusion units among the three groups of thalassemia patients. Likely, the regulation of hepcidin in thalassemia patients is affected more by erythropoietic activity than iron storage.     

An Enzyme That Inactivates the Inflammatory Mediator Leukotriene B4 Restricts Mycobacterial Infection

While tuberculosis susceptibility has historically been ascribed to failed inflammation, it is now known that an excess of leukotriene A₄ hydrolase (LTA4H), which catalyzes the final step in leukotriene B₄ (LTB₄) synthesis, produces a hyperinflammatory state and tuberculosis susceptibility. Here we show that the LTB₄-inactivating enzyme leukotriene B₄ dehydrogenase/prostaglandin reductase 1 (LTB4DH/PTGR1) restricts inflammation and independently confers resistance to tuberculous infection. LTB4DH overexpression counters the susceptibility resulting from LTA4H excess while ltb4dh-deficient animals can be rescued pharmacologically by LTB₄ receptor antagonists. These data place LTB4DH as a key modulator of TB susceptibility and suggest new tuberculosis therapeutic strategies.     

Single Low Dose Primaquine (0.25mg/kg) Does Not Cause Clinically Significant Haemolysis in G6PD Deficient Subjects

Background

Primaquine is the only drug consistently effective against mature gametocytes of Plasmodium falciparum. The transmission blocking dose of primaquine previously recommended was 0.75mg/kg (adult dose 45mg) but its deployment was limited because of concerns over haemolytic effects in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD deficiency is an inherited X-linked enzymatic defect that affects an estimated 400 million people around the world with high frequencies (15–20%) in populations living in malarious areas. To reduce transmission in low transmission settings and facilitate elimination of P. falciparum, the World Health Organization now recommends adding a single dose of 0.25mg/kg (adult dose 15mg) to Artemisinin-based Combination Therapies (ACTs) without G6PD testing. Direct evidence of the safety of this low dose is lacking. Adverse events and haemoglobin variations after this treatment were assessed in both G6PD normal and deficient subjects in the context of targeted malaria elimination in a malaria endemic area on the North-Western Myanmar-Thailand border where prevalence of G6PD deficiency (Mahidol variant) approximates 15%.

Methods and Findings

The tolerability and safety of primaquine (single dose 0.25 mg base/kg) combined with dihydroartemisinin-piperaquine (DHA-PPQ) given three times at monthly intervals was assessed in 819 subjects. Haemoglobin concentrations were estimated over the six months preceding the ACT + primaquine rounds of mass drug administration. G6PD deficiency was assessed with a phenotypic test and genotyping was performed in male subjects with deficient phenotypes and in all females. Fractional haemoglobin changes in relation to G6PD phenotype and genotype and primaquine round were assessed using linear mixed-effects models. No adverse events related to primaquine were reported during the trial. Mean fractional haemoglobin changes after each primaquine treatment in G6PD deficient subjects (-5.0%, -4.2% and -4.7%) were greater than in G6PD normal subjects (0.3%, -0.8 and -1.7%) but were clinically insignificant. Fractional drops in haemoglobin concentration larger than 25% following single dose primaquine were observed in 1.8% of the population but were asymptomatic.

Conclusions

The single low dose (0.25mg/kg) of primaquine is clinically well tolerated and can be used safely without prior G6PD testing in populations with high prevalence of G6PD deficiency. The present evidence supports a broader use of low dose primaquine without G6PD testing for the treatment and elimination of falciparum malaria.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01872702","term_id":"NCT01872702"}}NCT01872702     

Aptamer-graphene oxide for highly sensitive dual electrochemical detection of Plasmodium lactate dehydrogenase

A 90 mer ssDNA aptamer (P38) enriched against Plasmodium falciparum lactate dehydrogenase (PfLDH) through SELEX process was immobilized over glassy carbon electrode (GCE) using graphene oxide (GO) as an immobilization matrix, and the modified electrode was investigated for detection of PfLDH. The GO was synthesized from powdered pencil graphite and characterized by XRD based on the increased interlayer distance between graphitic layers from 0.345 nm for graphite to 0.829 nm for GO. The immobilization of P38 on GO was confirmed by I_D/I_G intensity ratio in Raman spectra where, the ratio were 0.67, 0.915, and 1.35 for graphite, GO and P38-GO, respectively. The formation of the P38 layer over GO-GCE was evident from an increase in the surface height in AFM analysis of the electrode from ∼3.5 nm for GO-GCE to ∼27 nm for P38-GO-GCE. The developed aptasensor when challenged with the target, a detection of as low as 0.5 fM of PfLDH was demonstrated. The specificity of the aptasensor was confirmed through a voltametric measurement at 0.65 V of the reduced co-factor generated from the PfLDH catalysis. Studies on interference from some common proteins, storage stability, repeatability and analysis of real samples demonstrated the practical application potential of the aptasensor.