Strong Anion Exchange Chromatography Research Articles

Chromatographic purification of C-Phycocyanin from Spirulina platensis: assessing antioxidant activity and stability.

Efficient separation and purification of proteins like C-phycocyanin (C-PC) from Spirulina platensis are essential for their commercialization, yet remain challenging. This study investigates three chromatographic methods: weak anion exchange chromatography (DEAE), strong anion exchange chromatography (Q Sepharose), and hydrophobic interaction chromatography (HIC) for C-PC purification. DEAE chromatography achieved a recovery of 36.80 mg unit (57.08%) with a purity of 3.23, outperforming Q Sepharose (yield: 23.21 mg unit means that 46.33%, purity: 2.76) and HIC (yield: 22.95 mg unit means that 17.57%, purity: 3.02). The purified C-PC consisted of α and β subunits with molecular masses of 16 kDa and 17 kDa, respectively. Further assessment revealed its antioxidant capacity through an ABTS assay. C-PC stability was tested at different pH levels and temperatures. Maximum stability was observed at pH 7, while pH 4 showed the lowest stability. Glutaraldehyde-treated C-PC (GC-PC) demonstrated gradual degradation up to 50°C, retaining 73.25% after 30 min. Notably, GC-PC exhibited stability even at higher temperatures, with degradation rates of 57.32% at 70°C and 50.96% at 80°C. DEAE chromatography proved superior for C-PC purification, offering higher yields and purity compared to Q Sepharose and HIC. The purified C-PC showed promising antioxidant capacity and stability, particularly GC-PC, which exhibited resistance to degradation even at elevated temperatures. These findings underscore the potential of C-PC as a valuable compound for various applications, with DEAE chromatography being an efficient method for its production and commercialization. This article is protected by copyright. All rights reserved.

Scale-Up of the Fermentation Process for the Production and Purification of Serratiopeptidase Using Silkworm Pupae as a Substrate.

Serratiopeptidase, a bacterial metalloprotease known for its pain-relieving and anti-inflammatory properties, can be produced through fermentation with S. marcescens. This study aimed to identify key factors related to nutrient composition and physicochemical conditions for production in Erlenmeyer flasks and to scale up the mixture to a bioreactor to obtain the maximum proteolytic activity. A Plackett-Burman design was used to determine whether the presence of silkworm pupae (at 1.5%) was a significant parameter for serratiopeptidase production. Along with the variables pH, temperature, and time, they were optimized using a Taguchi experimental design, resulting in values of 7, 25 °C, and 36 h, respectively. Scaling up with a kLa of 25.45 ± 3.12 h-1 showed the highest serratiopeptidase production at 24 h. A factorial design was used for ultrafiltration, resulting in an LMH (liters per square meter per hour) of 960 L/m2h, a TMP (transmembrane pressure) of 15 psi, and a concentration factor of five, with a specific activity of 24,325.81 ± 1515.69 U/mg. Afterward, the retentate was purified using strong anion exchange chromatography and ultrafiltration, yielding a 19.94 ± 3.07% recovery and a purification factor of 1.59 ± 0.31. In conclusion, waste from the sericulture industry can be used for serratiopeptidase production.

Maximal performance of intact N-glycopeptide enrichment using sequential HILIC and MAX columns.

Low abundance and heterogeneity of N-glycosylation at the peptide level poses a great challenge to the structural and functional analysis of glycosylation in the field of glycobiology. Solving this conundrum requires a sufficient and specific method for intact N-glycopeptide enrichment. Using the C18 or HLB desalting column followed by the mixed-mode strong anion exchange (MAX) or hydrophilic interaction chromatography (HILIC) glycopeptide enrichment column are commonly applied approaches for sample preparation of intact N-glycopeptides from complex samples. Herein, we compared the effects of different combinations of two desalting columns and two enrichment columns using equal amounts of mouse brain tissues from the same source. The results revealed the C18 column was a bit superior to the HLB column, and the MAX and HILIC columns were complementary on intact N-glycopeptides enrichment. Additionally, the results also demonstrated that enriching glycopeptides using a HILIC column followed by a MAX column from the flow-through solution got a better enrichment performance than the reversed order. Based on these results, the sequential enrichment of glycopeptides using HILIC and then MAX columns could maximize the enrichment performance of intact N-glycopeptides, and therefore is an option for in-depth analysis of site-specific glycoproteome.

Excretion of oxidated cortisol metabolites is markedly lower than previously assumed: An analysis of urinary cortoic acids in healthy children by GC–MS

Discovered about 50 years ago, the four C21 steroidal acids (α-)cortolic acid, β-cortolic acid, (α‑)cortolonic acid and β-cortolonic acid present the oxidative end products of cortisol metabolism. Undergoing renal elimination, these cortoic acids have been assumed to constitute up to 25 % of total urinary cortisol metabolites. However, their analysis has been difficult, only few data has been published in adults, and this class of steroids has become practically forgotten. Since data in children are lacking and nothing is known about their metabolism during human development, we aimed at establishing a more practical analytical method and determined their urinary concentrations in a high number of healthy subjects. In our method, 5-mL-aliquots of 24-hour urine samples were subjected to solid phase extraction (C18 cartridges), followed by strong anion exchange chromatography, and formation of 2-propylester-trimethylsilylether derivatives (2-PR/TMS). The cortoic acids were quantified by targeted gas chromatography–mass spectrometry (GC–MS) using a nonpolar GC column and selected ion monitoring (SIM). Baseline separation of all cortoic acids was achieved. Calibration graphs were linear (R2 > 0.98). Variations in precision and accuracy were less than 15 %, respectively. The detection limit was 100 pg (injected) with a signal-to-noise ratio of 3. 240 specimens from 24-hour urine collections from healthy children (120 boys, 120 girls, aged 3–18 years; DONALD study) were analyzed for cortoic acids and neutral cortisol metabolites to create first reference ranges. The profile of cortoic acids was dominated by α-cortolonic acid with excretion rates up to 70 µg/d. Absolute excretion rates of cortoic acids increased with age, their total excretion rates ranged between 11.0 and 127.3 µg/d (median 45.7 µg/d), but did not show any sexual dimorphism. Since cortoic acids make up only about 1 % of total urinary cortisol metabolites, determination of neutral urinary steroids reliably allows assessment of cortisol production. However, cortoic acids might present potential biomarkers of the body’s redox state.

Design of Experiment (DoE) for Optimization of HPLC Conditions for the Simultaneous Fractionation of Seven α-Amylase/Trypsin Inhibitors from Wheat (Triticum aestivum L.)

Wheat alpha-amylase/trypsin inhibitors remain a subject of interest considering the latest findings showing their implication in wheat-related non-celiac sensitivity (NCWS). Understanding their functions in such a disorder is still unclear and for further study, the need for pure ATI molecules is one of the limiting problems. In this work, a simplified approach based on the successive fractionation of ATI extracts by reverse phase and ion exchange chromatography was developed. ATIs were first extracted from wheat flour using a combination of Tris buffer and chloroform/methanol methods. The separation of the extracts on a C18 column generated two main fractions of interest F1 and F2. The response surface methodology with the Doehlert design allowed optimizing the operating parameters of the strong anion exchange chromatography. Finally, the seven major wheat ATIs namely P01083, P17314, P16850, P01085, P16851, P16159, and P83207 were recovered with purity levels (according to the targeted LC-MS/MS analysis) of 98.2 ± 0.7; 98.1 ± 0.8; 97.9 ± 0.5; 95.1 ± 0.8; 98.3 ± 0.4; 96.9 ± 0.5, and 96.2 ± 0.4%, respectively. MALDI-TOF-MS analysis revealed single peaks in each of the pure fractions and the mass analysis yielded deviations of 0.4, 1.9, 0.1, 0.2, 0.2, 0.9, and 0.1% between the theoretical and the determined masses of P01083, P17314, P16850, P01085, P16851, P16159, and P83207, respectively. Overall, the study allowed establishing an efficient purification process of the most important wheat ATIs. This paves the way for further in-depth investigation of the ATIs to gain more knowledge related to their involvement in NCWS disease and to allow the absolute quantification in wheat samples.

Versatile separation of nucleotides from bacterial cell lysates using strong anion exchange chromatography

Nucleotides exemplify some of the building blocks of life, comprising DNA & RNA, participating in processes such as cell signaling and metabolism, and serving as carriers of metabolic energy. The quantification of these compounds in biological samples is critical for researchers to understand complex systems. Herein, we demonstrate an anion exchange chromatography method utilizing a pH range of 8 to 10, which provides superior resolution and selectivity to previously reported methods and, more importantly, gives the flexibility to shift analyte selectivity if resolution between analytes is not optimal. We have applied the method to study the kinetics of the nucleotide pool in a bacterial cell-free lysate system that is producing RNA. Sample to sample runtimes are less than 18 min and recoveries greater than 96% were observed for all analytes through our methanol quench protocol with day-to-day variabilities less than 5%. This method reliably detects and quantifies all analytes that were expected to be observed in the process and helps lay the groundwork for future nucleotide research.

Highly sialylated mucin-type glycopeptide from porcine intestinal mucosa after heparin extraction: O-glycan profiling and immunological activity evaluation.

Mucins are the major proteins that distributed on the intestinal mucosa layer and protect the intestine from pathogens infection. The composition of intestinal mucin O-glycans can affect the health of the gastrointestinal tract in pigs. Porcine intestinal mucosa is widely used as the main raw material of heparin extraction. The heparin extraction residues rich in mucins were usually wasted. The structure of mucin derived O-glycans in porcine intestinal mucosa are currently unknown. In this study, we isolated the mucins from the heparin extraction residues and profiled the O-glycans. After heparin extraction, mucin was digested with trypsin, and separated by strong anion exchange chromatography. The mucin derived O-glycans were release by alkaline β elimination, and analyzed by ultra high performance liquid chromatography-porous graphitized carbon-Fourier transform mass spectrometry (UPLC-PGC-FTMS/MS). Thirty five kinds of O-glycans were identified, most of which were Core 3-derived glycans. In particular, the O-glycans containing sialic acid Neu5Ac accounted for 71.93% of the total O-glycans, which were different from that of other species, including mouse intestine, fish intestine, and porcine colon. The high content sialylated mucin may explain its effect in biological processes. Furthermore, the immunological activity results indicated that the porcine intestinal mucin could promote phagocytosis and proliferation without any cytotoxic effects, which may aid in the development of immunomodulators.

Two Dimensional Anion Exchange-Size Exclusion Chromatography Combined with Mathematical Modeling for Downstream Processing of Foot and Mouth Disease Vaccine

The production of high-quality purified virus particles in high quantities for vaccine preparation requires a scalable purification procedure in the downstream step. A purification scheme based on combined strong anion-exchange and size exclusion chromatography (2D-AEC-SEC) was developed for the production of non-structural protein-free foot and mouth disease vaccine, and the whole procedure was accomplished with 77.9% virus yield. Additionally, a mathematical modeling and a simulation approach based on a plate model of chromatography were developed and matched with the experimental chromatography data to improve prediction of retention behavior and save time in the development of the downstream scale-up method. The purified pooled virus fraction obtained from the final polishing step had a purity higher than 85% based on analytical size exclusion analysis. Moreover, more than 90.1% of residual DNA (rDNA) was removed from the purified vaccine. The analysis of purified virus particles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS), high performance size exclusion chromatography (HP-SEC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and transmission electron microscopy (TEM) provided clear evidence of purity and demonstrated that the final product is structurally spherical, intact particles qualified for formulation as a vaccine product.

Comprehensive analysis of heparinase derived heparin-products using two-dimensional liquid chromatography coupled with mass spectrometry

Heparin is a linear sulfated polysaccharide. It is composed of a repeating disaccharide unit with different sulfo patterns. The compositional analysis after heparin was decomposed to disaccharides and enzyme resistant domains is an important way to delve into its structure. Strong anion exchange (SAX) chromatography is commonly used for the compositional analysis due to its high resolution, stability and capability of quantitation. However, nonvolatile salt in mobile phase is not compatible with MS, then the structural domains cannot be identified without standards. Here, a new two-dimensional liquid chromatography system, multiple heart cut (MHC), was developed and linked to mass spectrometry (MS) directly to provide a comprehensive analysis of enzyme digested heparin. SAX was applied as the first dimensional chromatography, in which 17 peaks were observed and integrated in the digested heparin. Size-exclusion chromatography (SEC) was used as the second dimensional chromatography to desalt efficiently. Structural information of each component was then obtained with MS, including eight common disaccharides, eight enzyme resistant tetrasaccharides and a heparin–core protein linkage domain. The comparison of enzyme digested heparins obtained from different vendors using this system suggested their similar major structure and activity, but slightly different production processes.

Enrichment of Intact Glycopeptides Using Strong Anion Exchange and Electrostatic Repulsion Hydrophilic Interaction Chromatography.

Glycosylation is a biologically important and complex protein posttranslational modification. The emergence of glycoproteomic technologies to identify and characterize glycans on proteins has the potential to enable a better understanding the role of glycosylation in biology, disease states, and other areas of interest. In particular, the analysis of intact glycopeptides by mass spectrometry allows information about glycan location and composition to be ascertained. However, such analysis is often complicated by extensive glycan diversity and the low abundance of glycopeptides in a complex mixture relative to nonglycosylated peptides. Enrichment of glycopeptides from a protein enzymatic digest is an effective approach to overcome such challenges. In this chapter, we described a glycopeptide enrichment method combining strong anion exchange, electrostatic repulsion, and hydrophilic interaction chromatography (SAX-ERLIC). Following enzymatic digestion of proteins into peptides, SAX-ERLIC is performed by solid phase extraction to enrich glycopeptides from biological samples with subsequent LC-MS/MS analysis. Glycopeptide data generated using the SAX-ERLIC enrichment yields a high number of total and unique glycopeptide identifications which can be mapped back to proteins. The enrichment strategy is robust, easy to perform, and does not require cleavage of glycans prior to LC-MS/MS analysis.

Specific Non-Reducing Ends in Heparins from Different Animal Origins: Building Blocks Analysis Using Reductive Amination Tagging by Sulfanilic Acid.

Heparins are linear sulfated polysaccharides widely used as anticoagulant drugs. Their nonreducing-end (NRE) has been little investigated due to challenges in their characterization, but is known to be partly generated by enzymatic cleavage with heparanases, resulting in N-sulfated glucosamines at the NRE. Uronic NRE (specifically glucuronic acids) have been isolated from porcine heparin, with GlcA-GlcNS,3S,6S identified as a porcine-specific NRE marker. To further characterize NRE in heparinoids, a building block analysis involving exhaustive heparinase digestion and subsequent reductive amination with sulfanilic acid was performed. This study describes a new method for identifying heparin classical building blocks and novel NRE building blocks using strong anion exchange chromatography on AS11 columns for the assay, and ion-pair liquid chromatography-mass spectrometry for building block identification. Porcine, ovine, and bovine intestine heparins were analyzed. Generally, NRE on these three heparins are highly sulfated moieties, particularly with 3-O sulfates, and the observed composition of the NRE is highly dependent on heparin origin. At the highest level of specificity, the isolated marker was only detected in porcine heparin. However, the proportion of glucosamines in the NRE and the proportion of glucuronic/iduronic configurations in the NRE uronic moieties greatly varied between heparin types.

A new strategy of studying protein-protein interactions: Integrated strong anion exchange/reversed-phase chromatography/immunoprecipitation coupled with mass spectrometry for large-scale identification of proteins interact with immunoglobulin G in HeLa cells.

Recently, significant research efforts have been devoted to the development of technology for large-scale analysis of protein-protein interactions. Herein, a comprehensive method by coupling the first-dimension strong anion exchange chromatography with the second-dimension reversed-phase liquid chromatography via immunoprecipitation technique and high-resolution mass spectrometry analysis was developed for analyzing protein-protein interactions. After two-dimensional liquid chromatography separation, 108 fractions were obtained in one experiment. Immunoglobulin G from human serum was used as a model of an interacting protein. As a result, 919 proteins in these fractions were identified to interact with immunoglobulin G. By searching STRING database and data analysis, 27 of 919 proteins were inferred to directly interact with immunoglobulin G. Moreover, important target proteins that interacted with immunoglobulin G were mapped in the two-dimensional liquid chromatography system, which facilitated selection of these proteins from specific fractions. These results demonstrated that the proposed method can be useful for large-scale investigation of protein-protein interactions and as an advanced tool for the isolation of target proteins.

Purification, characterisation and antioxidant activities of chondroitin sulphate extracted from Raja porosa cartilage

This study was designed to isolate (isolation yield of 36.51 %) and characterise chondroitin sulphate (CS) from skate (Raja porosa) cartilage. Gel permeation chromatography demonstrated that the Raja porosa chondroitin sulphate (RPCS) obtained was a relatively uniform polysaccharide with a molecular weight of 40,752 Da and a purity of 94.0 %. Fourier-transform infrared, nuclear magnetic resonance spectroscopy and strong anion-exchange high-performance liquid chromatography indicated that the content of the predominant GlcA-GalNAc6S unit in RPCS was 65.84 %, which was higher than that in shark CS (53.93 %). Furthermore, RPCS displayed more effective free radical scavenging effects than did shark CS, indicating the potential of RPCS to promote oxidative stress resistance and to act as an antioxidant agent. Skate cartilage could be exploited as a sufficient and sustainable source for the preparation of CS with higher 6S-sulphation, which in turn could be scaled up for use in the pharmaceutical industry.

Chondroitin sulfate and hydroxyapatite from Prionace glauca shark jaw: Physicochemical and structural characterization

In the present work, the potential of the Prionace glauca jaw as a source of both chondroitin sulfate and bioapatite is explored. The sandwich-type structure in cross section of the jaw based on alternate layers with prevalence in organic tissue or mineralized is shown and these bands respectively confirmed as CS or hydroxyapatite -enriched zones. As result of this, an optimized process in sequential steps for the recovery of both biomaterials and their purification process is proposed, by combining enzymatic proteolysis, chemical precipitation and separation using ultrafiltration membrane for CS production together with controlled thermal treatment for hydroxyapatite obtaining. The purified CS was characterized by Gel Permeation Chromatography, Nuclear Magnetic Resonance and Strong Anion Exchange Chromatography, revealing a polymeric material with a molecular weight of 67 kDa, and prevalent 6S-GalNAc sulfation (68%), followed by 4S-GalNAc (13%), a significant proportion of disulfated disaccharides (12%) and only 7% of non-sulfated units. In the case of the bioapatite a purified biphasic 60:40 porous calcium phosphate of hydroxyapatite: whitlockite/β-TCP was confirmed. Hydroxyapatite as major component (85%) was also obtained for jaws directly subjected to the thermal treatment. This proved the influence of the enzymatic hydrolysis and centrifugation on the composition of the mineral fraction.

Biomedical copper speciation in relation to Wilson’s disease using strong anion exchange chromatography coupled to triple quadrupole inductively coupled plasma mass spectrometry

Biomedical analytical methods often rely on indirect measurements, such as immunoassays, which can lack effective metrological traceability. In the nephelometric determination of ceruloplasmin (Cp), an important protein whose circulating level is altered in Wilson’s disease (WD), the anti-Cp antibody used is not specific for the biologically active holoprotein so the assay can overestimate the concentration of Cp due to the presence of the apoprotein. By providing quantitation using elemental standards, the use of strong anion exchange chromatography (SAX) coupled to triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS-MS) can overcome the drawbacks of methods for the measurement of metalloproteins reliant on immunoassays. In the current study, a SAX-ICP-MS-MS method for Cp quantification was designed and tested in samples of blood serum of WD patients and healthy controls. Using standards based on a copper-EDTA complex for calibration, the method provides relatively simple quantification of Cp with the limit of detection of 0.1 μg L−1 (limit of quantification 0.4 μg L−1). The method was also used to investigate the copper species separated by using a 30 kDa cut-off ultrafiltration device. The so-called "exchangeable" copper fraction is considered as an alternative clinical biomarker of WD. Using the designed speciation approach, it was shown that the ultrafiltration method can overestimate the "exchangeable" copper fraction due to a removal of copper from Cp. This was confirmed by comparing the enzymatic activity of the fractions. Thus, the specificity of the "exchangeable" copper test can be ensured only under strict maintenance of ultrafiltration conditions.

Scalable Chemoenzymatic Synthesis of Inositol Pyrophosphates.

The inositol pyrophosphates (PP-InsPs) are an important group of cellular messengers that influence a broad range of biological processes. To elucidate the functions of these high-energy metabolites at the biochemical level, access to the purified molecules is required. Here, a robust and scalable strategy for the synthesis of various PP-InsPs [5PP-InsP5, 1PP-InsP5, and 1,5(PP)2-InsP4] is reported, relying on the highly active inositol hexakisphosphate kinase A from Entamoeba histolytica and the kinase domain of human diphosphoinositol pentakisphosphate kinase 2. A facile purification procedure using precipitation with Mg2+ ions and an optional strong anion exchange chromatography on an FPLC system afforded PP-InsPs in high purity. Furthermore, the newly developed protocol could be applied to simplify the synthesis of radiolabeled 5PP-InsP5-β32P, which is a valuable tool for studying protein pyrophosphorylation. The chemoenzymatic method for obtaining PP-InsPs is readily amenable to both chemists and biologists and will thus foster future research on the multiple signaling functions of PP-InsP molecules.

Strong anion exchange‐mediated phosphoproteomics reveals extensive human non‐canonical phosphorylation

Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site‐specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other “non‐canonical” amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non‐canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry‐based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non‐canonical phosphosites is approximately one‐third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high‐throughput exploration of non‐canonical phosphorylation in all organisms.

Development of a New Tandem Ion Exchange and Size Exclusion Chromatography Method To Monitor Vaccine Particle Titer in Cell Culture Media.

A new tandem chromatography method was developed to directly measure the titers of various vaccine candidate molecules in cell culture without a prior purification step. The method utilized a strong anionexchangechromatography (IEC) column in tandem with a sizeexclusionchromatography (SEC) column to efficiently separate the nanoparticle and virus-like particle (VLP) vaccine molecules from host cell proteins and other components in the cell culture media. The dual (charge and hydrodynamic size) separation mode was deemed necessary to achieve good separation of the vaccine product for quantitation. The method development and quality assessment illustrated herein was focused on the influenza vaccine candidate H1ssF, a hemagglutinin (group 1) stabilized stem molecule fused to ferritin to form nanoparticles. This newly established method was then successfully applied to several vaccine candidate developmental projects, such as the hemagglutinin-ferritin (HAF) nanoparticle and encephalitic alphavirus VLP-based vaccines. This IEC-SEC strategy was established as a platform approach for direct titer measurement of novel vaccine molecules in cell culture.

Sulphated Polysaccharide from Acanthophora spicifera Induced Hevea brasiliensis Defense Responses Against Phytophthora palmivora Infection.

Elicitors from seaweeds are considered an alternative stimulant of plant defenses against pathogenic infection. Finding new sources of elicitors and exploring their effects on plant defenses is a significant undertaking. In this study, we extracted crude polysaccharide (CPS) from Acanthophora spicifera (a red alga) and tested the effects of the compound on rubber tree (Hevea brasiliensis) defense responses. Accumulations of salicylic acid (SA) and scopoletin (Scp) were measured by HPLC. The expression of SA- and Jasmonic acid (JA)-responsive genes was analyzed by semi-qRT-PCR. Strong anion exchange chromatography and Fourier-transform infrared (FTIR) spectroscopy were used for purification and functional characterization of CPS, respectively. The extracted CPS enhanced rubber tree defenses against Phytophthora palmivora infection. It induced SA and Scp accumulations and SA-responsive gene expression, but suppressed JA-responsive gene expression. We successfully separated the non-sulphated polysaccharide (F1) from the sulphated polysaccharides (SPS). Both peaks of SPS (F2 and F3) were identified as lambda (λ)-carrageenan. The F3 fraction showed greater elicitor activity on tobacco leaves. It induced SA and Scp accumulations and peroxidase activity but suppressed catalase activity. Furthermore, the purified λ-carrageenan did not cause cell death in tobacco or rubber tree leaves. Therefore, the elicitor from A. spicifera could be an alternative plant stimulant.

Elimination of ghost peaks by optimization of anion exchange chromatography method for determination of gamma-carboxyglutamic acid (Gla)-domainless impurity in recombinant activated clotting factor VII drug products

Secreted recombinant activated clotting factor VII activated (rFVIIa) in cell culture media missing gamma-carboxyglutamic acid (Gla) domain as a result of failure in gamma-carboxylation or cell lysis is called Gla-domainless impurity which has less negative charge compared to native rFVIIa. Based on risk assessment, this type of impurity is considered as critical drug product quality attribute of rFVIIa and its quantitative analysis in product batches is a critical issue in quality control laboratories.Analysis of Gla-domainless impurity is accomplished by Strong Anion Exchange Chromatography (SAX) in recombinant factor VIIa using Tris and Bis-Tris propane salt buffers as equilibrating buffers and high concentration ammonium acetate as an eluent. Appearance of ghost peaks with notable intensity during elution time of Gla-domainless impurity caused distortion of the related peak and interference with robust and accurate quantification of this impurity. Subsequently, the ghost peak was analyzed by LC-ESI-MS to determine the structure which showed the m/z values at 905.27, 623.53 and 341.60 and 563.73.To find the source of these ghost peaks, quality of water, buffer salts and Chelex-100 together with ionic strength of mobile phase A (addition of 25 mM NaCl) were considered as affecting parameters and several experiments designed with DOE software to optimize the best condition of highest quality the method with lowest signal of ghost peak noises.By interpretation of DOE result, it is concluded that high grade water and buffer salt along with high quality Chelex-100 resins are important factors to achieve a method with lowest ghost peaks. However, addition of 25 mM NaCl to mobile phase A with either lower quality buffer salts or lower water grade yields high quality chromatogram peak with acceptable ghost peaks. LC/MS analysis indicates that macrostructures of Bis-Tris propane made up as a result of hydrogen bonds with each other or Tris molecules can be the source of ghost peaks.