Complex Elution Profiles Research Articles

Application of the Steric Mass Action formalism for modeling under high loading conditions: Part 2. Investigation of high loading and column overloading effects

The application of a model-based approach for industrial chromatography development requires the capability of the model to describe protein elution under high loading and overloading conditions. In a previous work, an extensive dataset was created to model the elution behavior of a bispecific antibody (bsAb) on the strong cation exchange resin POROS™ XS. Thereby, the pH-dependence of the model parameters in the Steric Mass Action (SMA) model could be examined and described over a pH range of 4.5 to 8.9. However, discrepancies between simulated and experimental data were observed under high loading and overloading conditions, particularly in the lower pH range (pH 4.5 to 5.3) and in the higher pH range (pH 6.0 to 9.0). In this work, these discrepancies are studied by performing new experiments which show that these differences were primarily not caused by limitations of the SMA model. At lower pH values, overloading phenomena such as protein breakthrough during the loading phase, additional peaks, and peak shoulders occurred. The application of various experiments performed with different Na+ concentrations and different loading times during sample loading revealed that intraparticle diffusion effects and conformational changes of the bsAb are responsible for these overloading phenomena at low pH. The applied lumped rate mass transfer model is not adequate and should be extended to consider these effects. At higher pH, the assumption of describing the bsAb's elution behavior with only one simulated species was insufficient to predict complex peak shapes that arise because of multi-component elution of the bsAb's charge variants. The extension of the model to a simple multi-component system consisting of two variants allowed the prediction of a majority of the complex elution profiles.

Purification of lectin and Kunitz trypsin inhibitor from soya seeds.

The search for potent and selective therapeutic agents is progressing by the study of natural compounds in plants. Plant-derived macromolecules are considered emerging therapeutic agents and an alternative to synthetic and small molecule drugs. Where it has long been known that plants possess medicinal properties, the compounds responsible for their action are in many cases still unknown: often only whole crude plant extracts or fractionated extracts are tested for the ability to inhibit common pathogens. Here, we present a fast protein liquid chromatography method for the separation of crude plant proteins. Kunitz trypsin inhibitor (KTI; 24.2 kDa) and lectin (31 kDa) were purified from Glycine max by liquid extraction followed by ion exchange column chromatography. The need for serial chromatographic separation steps has been eliminated by introducing more complex elution profiles hence reducing cost, time and improving recovery. The identity of KTI-A and lectin was confirmed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-ToF MS). Cell proliferation assays using B16F1 melanoma cells revealed that both KTI and the monomeric lectin retained some antiproliferative activity. This method could be useful for rapid and cost-effective purification of bioactive compounds from plant material.

Computer-assisted multi-segment gradient optimization in ion chromatography

This study reports simulation and optimization of ion chromatography separations using multi-segment gradient elution. First, an analytical expression for the gradient retention factor under these complex elution profiles was derived. This allows a rapid retention time prediction calculations under different gradient conditions, during computer-assisted method development. Next, these analytical expressions were implemented in an in-house written Matlab® routine that searches for the optimal (multi-segment) gradient conditions, either via a four-segment grid search or via the recently proposed one-segment-per-component search, in which the slope is adjusted after the elution of each individual component. Evaluation of the retention time simulation and optimization approaches was performed on a mixture of 18 inorganic anions and different subsets with varying number of compounds. The two considered multi-segment gradient optimization searches resulted in similar proposed gradient profiles, and corresponding chromatograms. Moreover, the resultant chromatograms were clearly superior to the chromatograms obtained from the best simple linear gradient profiles, found via a fine grid search. The proposed approach is useful for automated method development in ion chromatography in which complex elution profiles are often used to increase the separation power.

EComment. The devil is in the detail!

I read with great interest the quasi-experimental single-centre prospective cohort study conducted by Birgand et al. [1] evaluating the efficacy of a gentamicin-collagen sponge in decreasing deep sternal-wound infections in high-risk cardiac surgery patients. Interstingly, in their experience gentamicin-collagen sponge was not effective in preventing deep sternal wound infections in high-risk patients. This is contrary to our own experience [2] and the most plausible explanation for the conflicting results lies in the technique of using the gentamicin-collagen sponge. Birgand and associates dipped the sponge in normal saline solution for a few seconds prior to implantation [1]. This manoeuvre of wetting the gentamicin-collagen sponge impacts on the gentamicin content of the implant and affects its potential antibacterial efficacy. Lovering and colleague [3] have recently shown that that even a short period of dipping of gentamicin-collagen sponge, before insertion into the patient, results in a significant loss of gentamicin which may be of clinical significance. They demonstrated that after a very short immersion period there was significant loss of gentamicin from the implants with a mean loss of 6.7% at 2 s, increasing to 40.5% at 1 min and essentially total loss by 6 h of immersion. Loss of gentamicin followed a complex elution profile, with elution half-lives ranging from 50 s on initial immersion to 99 min late in the elution period. It is very clear from the study of Lovering et al. [3] as well as from our own experience [2] that attention to detail will possibly result in different outcomes thereby minimising studies with conflicting results. Conflict of interest: none declared

Determination of pharmaceutically related compounds by suppressed ion chromatography: IV. Interfacing ion chromatography with universal detectors

This work forms the final part of a study investigating gradient elution ion-exchange chromatography of pharmaceutically relevant compounds, aiming at achieving complementary selectivity to reversed-phase HPLC. In this study the coupling of three universal detectors (electro-spray ionisation mass spectrometer (ESI-MS); corona charged aerosol detector (CAD); and evaporative light scattering detector (ELSD)) to suppressed IC using complex elution profiles with potassium hydroxide eluents is demonstrated. The non-volatile ions were removed from the eluent by the suppressor prior to detection, thus allowing a stable detector response, especially with the prototype electrolytic suppressor. The detector response for ten weakly anionic pharmaceuticals followed the expected models and the limits of detection obtained were not compromised by the use of a suppressor, yielding values below 50ng/mL with MS, low to sub μg/mL levels with CAD and 2–20μg/mL with ELSD (25μL injections). When coupled to MS and CAD, the prototype electrolytic suppressor showed percentage relative standard deviations (%RSDs) in peak areas of 0.4–2.5% on average, compared to the chemical suppressor which yielded 1.5–3 fold higher %RSD values for the test analytes. The prototype electrolytic suppressor also generally exhibited wider linear response ranges than the chemical suppressor.

Impact of soaking gentamicin-containing collagen implants on potential antimicrobial efficacy

BackgroundThe purpose of this study is to evaluate how wetting of Collatamp (a gentamicin-containing collagen implant [GCCI]) impacts on the gentamicin content of the implant and whether this affects its potential antibacterial efficacy. MethodsGCCI (Collatamp®, EUSA Pharma [Europe], Oxford, United Kingdom) containing 130 mg gentamicin and 280 mg collagen (10 cm × 10 cm) were immersed in 300 mL normal saline for up to 6h. At set times after immersion the GCCI were removed, the saline diluted in normal human serum and the gentamicin content assayed by a validated immunoassay (Cedia, Microgenics Ltd, UK) to provide an estimate of the loss from each implant. The mean concentration data were then fitted to an exponential decay model (WinNonLin, Pharsight, US). ResultsAfter a very short immersion period there was significant loss of gentamicin from the implants with a mean loss of 6.7% at 2 s, increasing to 40.5% at 1 min and essentially total loss by 6 h of immersion. Loss of gentamicin followed a complex elution profile, with elution half-lives ranging from 50 s on initial immersion to 99 min late in the elution period. ConclusionThis study provides clear evidence that even a short period of dipping of Collatamp implants, and probably other GCCI, before insertion into the patient results in a significant loss of gentamicin which may be of clinical significance unless the period of soaking is very short. We therefore recommend that wetting of these implants before insertion is not undertaken.

Fractionation and Characterization of Completely Dissolved Ball Milled Hardwood

A simplified method for sequentially dissolving and fractionating ball milled hard wood polymers in high yield is presented. The complete dissolution of the whole ball milled wood, and the fractions recovered from it by applying the sequential fractionation, was accomplished in lithium chloride/dimethylacetamide solvent system enabling size exclusion chromatography studies. The chromatography applied a four detector system; an Ultraviolet-Visible spectroscopy-, Intrinsic Viscosity-Differential Pressure-, Right Angle Laser Light Scattering- and Refractive Index detectors, a combination hereby proposed to enable interpretation of the complex elution profiles with emphasis on chemical interactions. Indirect evidence of the presence of lignin carbohydrate complexes in hardwoods is provided. It is also proposed that cellulose is severely degraded from a degree of polymerization of 10,000 in wood, down to a value of approximately 400 by either the applied ball milling condition or in subsequent solubilization steps.

Methodology for porting retention prediction data from old to new columns and from conventional-scale to miniaturised ion chromatography systems

Several procedures are available for simulating and optimising separations in ion chromatography (IC), based on the application of retention models to an extensive database of analyte retention times on a wide range of columns. These procedures are subject to errors arising from batch-to-batch variability in the synthesis of stationary phases, or when using a column having a different diameter to that used when the database was acquired originally. Approaches are described in which the retention database can be recalibrated to accommodate changes in the stationary phase (ion-exchange selectivity coefficient and ion-exchange capacity) or in the column diameter which lead to changes in phase ratio. The entire database can be recalibrated for all analytes on a particular column by performing three isocratic separations with two analyte ions. The retention data so obtained are then used to derive a “porting” equation which is employed to generate the required simulated separation. Accurate prediction of retention times is demonstrated for both anions and cations on 2 mm and 0.4 mm diameter columns under elution conditions which consist of up to five sequential isocratic or linear gradient elution steps. The proposed approach gives average errors in retention time prediction of less than 3% and the correlation coefficient was 0.9849 between predicted and observed retention times for 344 data points comprising 33 anionic or cationic analytes, 5 column internal diameters and 8 complex elution profiles.

P.2.c.010 Comparison of BMI changes associated with various antidepressant treatments during acute depression

The purpose of this study is to evaluate how wetting of Collatamp (a gentamicin-containing collagen implant [GCCI]) impacts on the gentamicin content of the implant and whether this affects its potential antibacterial efficacy.GCCI (Collatamp®, EUSA Pharma [Europe], Oxford, United Kingdom) containing 130 mg gentamicin and 280 mg collagen (10 cm × 10 cm) were immersed in 300 mL normal saline for up to 6h. At set times after immersion the GCCI were removed, the saline diluted in normal human serum and the gentamicin content assayed by a validated immunoassay (Cedia, Microgenics Ltd, UK) to provide an estimate of the loss from each implant. The mean concentration data were then fitted to an exponential decay model (WinNonLin, Pharsight, US).After a very short immersion period there was significant loss of gentamicin from the implants with a mean loss of 6.7% at 2 s, increasing to 40.5% at 1 min and essentially total loss by 6 h of immersion. Loss of gentamicin followed a complex elution profile, with elution half-lives ranging from 50 s on initial immersion to 99 min late in the elution period.This study provides clear evidence that even a short period of dipping of Collatamp implants, and probably other GCCI, before insertion into the patient results in a significant loss of gentamicin which may be of clinical significance unless the period of soaking is very short. We therefore recommend that wetting of these implants before insertion is not undertaken.

Prediction of Analyte Retention for Ion Chromatography Separations Performed Using Elution Profiles Comprising Multiple Isocratic and Gradient Steps

This study addresses the simulation of ion chromatographic (IC) separations performed under conditions where the elution profile consists of a sequence of isocratic and gradient elution steps (referred to as "complex elution profiles"). First, models for prediction of retention under gradient elution conditions in IC were evaluated using an extensive database of gradient elution retention data. It is shown that one such model is preferred on the basis that it can be used to predict gradient retention times on the basis of isocratic input data. A method is then proposed for using this model for complex elution profiles whereby each step of the elution profile is treated separately and analyte movement through the column is mapped. An empirically based algorithm for predicting peak width under complex elution conditions is also proposed. Evaluation of the suggested approaches was undertaken on a set of 24 analyte anions and 13 analyte cations on 5 different Dionex columns using a range of 5-step complex elution profiles that gave R2 values for correlations between predicted and observed retention times of 0.987 for anions and 0.997 for cations. The simulation of separations of anions and cations using a 3-step complex elution profile is demonstrated, with good correlation between observed and predicted chromatograms. The proposed approach is useful for the rapid development of separations when complex elution profiles are used in IC.

The Use of Ancestral Haplotypes in the Molecular Diagnosis of Familial Breast Cancer

Mutations in BRCA1 and BRCA2 account for about 40% of families with an inherited susceptibility to breast and/or ovarian cancer. Mutational analysis of these two genes has become the standard of care for families with a strong suggestion of inherited susceptibility. Methodologies for screening vary, but one of the more popular techniques is dHPLC, due to its combination of high sensitivity and low cost. The presence of a large number of polymorphisms in the two BRCA genes complicates dHPLC analysis, often leading to complex elution profiles. There are concerns that a pattern produced by a sample heterozygous for a polymorphism may be very similar to that produced by a sample heterozygous for a unique mutation within the same amplicon. Further molecular analysis is often required to resolve whether any given shift is due to a polymorphism or a disease-causing mutation. The use of ancestral haplotypes was explored as a means to minimize the need for further analysis. Groups of 86 patients were genotyped for 12 BRCA1 polymorphisms or 20 BRCA2 polymorphisms. For BRCA1, eight distinct haplotypes were identified, which are largely derivatives of two main lineages. For BRCA2, 17 distinct haplotypes were identified, leading to a much more complex polymorphic pattern. With this knowledge, we have defined a system to determine which patients, if any, require further investigations. This method could be used to supplement any comprehensive screening methodology for other large genes that lie within strong regions of linkage disequilibrium such as NF1, CFTR, MLH1, or MSH2.

538 New synthetic Epothilone derivative ZK-EPO inhibits tumors generally resistant to chemotherapy

The purpose of this study is to evaluate how wetting of Collatamp (a gentamicin-containing collagen implant [GCCI]) impacts on the gentamicin content of the implant and whether this affects its potential antibacterial efficacy.GCCI (Collatamp®, EUSA Pharma [Europe], Oxford, United Kingdom) containing 130 mg gentamicin and 280 mg collagen (10 cm × 10 cm) were immersed in 300 mL normal saline for up to 6h. At set times after immersion the GCCI were removed, the saline diluted in normal human serum and the gentamicin content assayed by a validated immunoassay (Cedia, Microgenics Ltd, UK) to provide an estimate of the loss from each implant. The mean concentration data were then fitted to an exponential decay model (WinNonLin, Pharsight, US).After a very short immersion period there was significant loss of gentamicin from the implants with a mean loss of 6.7% at 2 s, increasing to 40.5% at 1 min and essentially total loss by 6 h of immersion. Loss of gentamicin followed a complex elution profile, with elution half-lives ranging from 50 s on initial immersion to 99 min late in the elution period.This study provides clear evidence that even a short period of dipping of Collatamp implants, and probably other GCCI, before insertion into the patient results in a significant loss of gentamicin which may be of clinical significance unless the period of soaking is very short. We therefore recommend that wetting of these implants before insertion is not undertaken.

Determination of interconversion barriers by dynamic gas chromatography: epimerization of chalcogran.

The four stereoisomers of chalcogran 1 ((2RS,SRS)-2-ethyl-1,6-di-oxaspiro[4.4]nonane), the principal component of the aggregation pheromone of the bark beetle pityogenes chalcographus, are prone to interconversion at the spiro center (C5). During diastereo- and enantioselective dynamic gas chromatography (DGC), epimerization of 1 gives rise to two independent interconversion peak profiles, each featuring a plateau between the peaks of the interconverting epimers. To determine the rate constants of epimerization by dynamic gas chromatography (DGC), equations to simulate the complex elution profiles were derived, using the theoretical plate model and the stochastic model of the chromatographic process. The Eyring activation parameters of the experimental interconversion profiles, between 70 and 120 C in the presence of the chiral stationary phase (CSP) Chirasil-beta-Dex, were then determined by computer-aided simulation with the aid of the new program Chrom-Win: (2R,5R)-1: deltaG(++) (298.15 K) = 108.0 +/-0.5 kJ mol(-1), deltaH(++) = 47.1+/-0.2 kJ mol(-1), deltaS(++) = -204+/-6 JK(-1) mol(-1): (2R,5S)-1: deltaG(++) (298.15 K) = 108.5+/-0.5 kJ mol(-1), deltaH(++) = 45.8+/-0.2 kJ mol(-1), deltaS(++) = -210 +/-6 J K mol(-1); (2S,5S)-1: deltaG(++) (298.15 K)= 108.1+/-0.5 kJ mol(-1), deltaH(++) = 49.3+/-0.3 kJ mol(-1), deltaS(++) = -197+/-8 J K(-1) mol(-1); (2S,5R)-1: deltaG(++) (298.15 K)=108.6+/-0.5 kJ mol(-1), deltaH(++) = 48.0+/-0.3 kJ mol(-1), deltaS(++) = -203+/-8 J K(-1) mol(-1). The thermodynamic Gibbs free energy of the E/Z equilibrium of the epimers was determined by the stopped-flow multidimensional gas chromatographic technique: deltaG(E/Z) (298.15 K)= -0.5 kJ mol(-1), deltaH(E/Z) = 1.4 kJ mol(-1) and deltaS(E/Z) = 6.3 J K(-1) mol(-1). An interconversion pathway proceeding through ring-opening and formation of a zwitterion and an enol ether/alcohol intermediate of 1 is proposed.

Analysis of Partially Methyl-Esterified Galacturonic Acid Oligomers by High-Performance Anion-Exchange Chromatography and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Two methods were developed to detect partially methyl-esterified galacturonic acid oligomers, generated by endopolygalacturonase treatment of a 30% methyl-esterified pectin. The enzyme digest was shown, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, to contain sodiated galacturonic acid oligomers with a degree of polymerization of 2–12, containing 0–6 methyl esters. Galacturonic acid (monomer) could not be detected because of matrix ions interference in the low mass region. Using high-performance anion-exchange chromatography, with a sodium acetate gradient at pH 5.0 and postcolumn sodium hydroxide addition to allow pulsed amplified detection, a complex elution profile was obtained with the endopolygalacturonase-treated 30% methyl-esterified pectin. All the components eluted before nonesterified tetragalacturonic acid. The partially methyl-esterified oligogalacturonic acids eluted in a discernible series of oligomers with an identical number of nonesterified carboxylic acid groups; the large, more esterified oligomers eluted before small, less esterified oligomers. The methyl esters may hinder the interaction of the neighboring carboxylic acid groups with the anion-exchange resin, thereby giving the components an apparent lower overall negative charge.

A neuropeptide Y (NPY)-related peptide is present in the river lamprey CNS

Pancreatic polypeptide (PP)-like material from brain+spinal cord, and retina extracts of Lampetra fluviatilis was studied by HPLC and RIA. The brain+spinal cord extract showed a complex elution profile with multiple peaks of immunoreactivity. The retina extract showed a much simpler pattern with a single significant peak along with a trace of a second peak corresponding to the latest and penultimate peaks in the brain extract. Twenty-one out of 36 residues could be sequenced from the latest eluting peak in the brain extract. This sequence showed 81% identity with porcine neuropeptide (NPY) suggesting that both the brain/spinal cord and retina of the river lamprey contain a peptide homologous to NPY.

Multiple forms of pancreatic polypeptide-related compounds in the lamprey CNS: partial characterization and immunohistochemical localization in the brain stem and spinal cord

Although neuropeptide Y (NPY) is established as a transmitter in many regions of the nervous system, the role of other peptides of the pancreatic polypeptide (PP) family in the CNS is obscure. This study provides evidence that PP-like peptides in the "primitive" CNS of a cyclostome are composed of different molecular forms, which are stored in separate neuronal populations with apparently different functions. PP-like material was detected in extracts of brain and spinal cord from Lampetra fluviatilis by radioimmunoassay (RIA) using an antiserum to the C-terminal hexapeptide of mammalian PP. The PP-immunoreactive material consisted of several molecular forms, as shown by its complex elution profile on high-performance liquid chromatography (HPLC). The cellular distribution of PP-like immunoreactivity was studied with indirect immunofluorescence histochemistry using antisera toward porcine peptide YY (PYY), porcine neuropeptide Y (NPY), and bovine (BPP), rat (RPP), and avian (APP) pancreatic polypeptide. Adjacent sections from the brain stem and spinal cord of L. fluviatilis and Ichthyomyzon unicuspis, incubated with the different antisera, displayed 2 main patterns of PP immunoreactivity. The PYY and RPP antisera labeled groups of neurons and fibers in the rhombencephalic and mesencephalic reticular formation. One of the PYY/RPP-ir cell groups, located in the anterior rhombencephalic reticular nucleus, had a projection to the dorsolateral spinal cord. Fibers of this reticulospinal system were in close apposition to dendrites of intracellularly stained spinal motoneurons and sensory relay interneurons, indicating that they may receive PPergic input. In contrast, antisera to NPY and APP labeled local neurons systems in the spinal dorsal horn, in the lateral parts of the brain stem, including the rhombencephalic alar plate, and in the retina. The BPP antiserum recognized the NPY/APP as well as the PYY/RPP immunoreactive neuron systems, further supporting that they both contain PP-like peptides.

Cell Cycle Effect on the Induction of DNA Double-Strand Breaks by X Rays

Filter elution was used to compare X-ray-induced DNA single- and double-strand breaks in proliferating (P) and quiescent (Q) cells of the 66 and 67 mouse mammary tumor lines. There was no difference either between cell type or between growth states in the amount of single-strand breaks as defined by elution at pH 12.2. In contrast, Q cells appeared to sustain a much larger amount of double-strand break damage per Gray than P cells, when the damage was measured by elution at either pH 7.2 or pH 9.6. Experiments which combined centrifugal elutriation with pH 7.2 elution demonstrated that G1-P cells were similar to Q (greater than or equal to 95% G1) cells in the induction of elution-detectable double-strand breaks, while the S-phase enriched fractions sustained less damage than G1-P, Q, or asynchronous P populations. Studies in which P populations were pulse labeled with [14C]thymidine confirmed this finding. Mathematical analysis of the elution kinetics of irradiated P, Q, and S-phase cells supports a model in which the complex elution profiles observed for P cells could be explained as the sum of the one-component exponential elution profiles of G1- and S-phase subpopulations. Also, the correlation between damage measured by pH 7.2 elution and cell survival was tested by examining the dose response for stimulated 66 cells (St4), which like Q cells are greater than or equal to 95% in G1 but are more resistant to X-ray-induced cytotoxicity than are the 66 Q cells. However, the induction of double-strand breaks in St4 cells was identical to that in Q cells. Thus we conclude that there is not necessarily a correlation between the amount of elution-detectable X-ray-induced double-strand breaks and cell survival.