Journal of Chromatography B (v.938, #C)
Editorial Board (i).
Highly specific separation for antitumor Spiropreussione A from endophytic fungal [Preussia sp.] fermentation broth by one-step macroporous resins AB-8 treatment by Bing Li; Chunlan Wang; Xiaomei Chen; Jing Lyu; Shunxing Guo (1-7).
It is attractive to pharmaceutical works to seek useful material from endophytic fungi. Spiropreussione A (SA) which is isolated from endophytic fungus Preussia sp. is a novel anti-tumor product. Since previous preparation method cannot provide enough SA, and considering the large volume of broth and low concentration of the target product, macroporous resins were introduced to separate SA in our study. Four kinds of macroporous resins ADS-8, H103, X-5 and AB-8 were applied for separating SA, and AB-8 was selected as the optimal resin according to its performances through static and dynamic measurements. HPLC was used to analyze SA in all samples. Under optimal conditions, the specific SA adsorption capacity of AB-8 resin was 15.23 mg/g, and the purity increased by 2.5-fold from 35.0% in broth to 90.0% in eluent with 70.0% recovery yield by a one-step treatment. Conclusively, our study achieved the goal of separating and purifying SA in high efficiency, and offered references for further fermentation works.
Keywords: Endophytic fungi; Fermentation broth; Spiropreussione A; Separation; Macroporous resin;
Determination of amantadine and rimantadine in chicken muscle by QuEChERS pretreatment method and UHPLC coupled with LTQ Orbitrap mass spectrometry by Hua Yan; Xin Liu; Fengyun Cui; Huan Yun; Jianhui Li; Shuangyang Ding; Dajin Yang; Zhaohui Zhang (8-13).
A novel sample pretreatment method was developed for the quantitative determination of amantadine and rimantadine in chicken muscle tissues by ultra high performance liquid chromatography coupled with high resolution LTQ Orbitrap mass spectrometry (UHPLC-LTQ Orbitrap MS). The samples were pretreated by modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method, using acetonitrile (1% acetic acid, v/v) as extraction solution and C18 sorbent for clean-up. The separation was carried out on a Waters ACQUITY UPLC HSS T3 column (150 mm × 2.1 mm, 1.8 μm particle), using a mobile phase of acetonitrile and 0.1% aqueous formic acid solution. LTQ Orbitrap MS with resolving power of 60 000 was applied for analysis of the samples. Amantadine and rimantadine were identified from their accurate mass (within 5 ppm) and retention times from the acquired full-scan chromatogram and quantified by their peak areas. The linear range for the determination of the analytes was 1–100 μg/kg. Limits of detection (LODs) for amantadine and rimantadine were 1.02 μg/kg and 0.67 μg/kg, respectively. The intra- and inter-day accuracy ranged from 87.5% to 102.4%, and 82.5% to 105.8% for amantadine, and 95.3% to 97.4%, and 89.4% to 93.2% for rimantadine, respectively. The precision of intra- and inter-day was between 3.9–6.3% and 5.95–13.9% for amantadine, 6.0–7.45% and 7.8–12.4% for rimantadine, respectively. Finally, the method was applied for the determination of these antiviral agents in routine samples, and amantadine residue was detected in some cases.
Keywords: Amantadine; Rimantadine; QuEChERS method; UHPLC; LTQ Orbitrap MS; Chicken muscle tissues;
Purification of antibodies by precipitating impurities using Polyethylene Glycol to enable a two chromatography step process by Glen Giese; Adam Myrold; Jeffrey Gorrell; Josefine Persson (14-21).
The purification of antibodies by precipitating impurities using Polyethylene Glycol (PEG) was assessed with the objective of developing a two chromatography column purification process. A PEG precipitation method was evaluated for use in the industrial purification of recombinant monoclonal antibodies (MAbs). Effective and robust precipitation conditions including PEG concentration, pH, temperature, time, and protein concentration were identified for several different MAbs. A recovery process using two chromatography steps in combination with PEG precipitation gave acceptable yield and purity levels for IgG1 and IgG4 antibodies with a broad range of isoelectric points (pI). PEG precipitation removed host cell proteins (HCPs), high molecular weight species (HMWS), leached Protein A ligand, and host cell DNA to acceptable levels when run under appropriate conditions, and some endogenous virus removal was achieved.
Keywords: Antibody; Purification; PEG; Precipitation;
Analysis of non-esterified fatty acids in human samples by solid-phase-extraction and gas chromatography/mass spectrometry by Thomas Kopf; Gerd Schmitz (22-26).
The determination of the fatty acid (FA) profile of lipid classes is essential for lipidomic analysis. We recently developed a GC/MS-method for the analysis of the FA profile of total FAs, i.e. the totality of bound and unbound FAs, in any given biological sample (TOFAs). Here, we present a method for the analysis of non-esterified fatty acids (NEFAs) in biological samples, i.e. the fraction that is present as extractable free fatty acids. Lipid extraction is performed according to Dole using 80/20 2-propanol/n-hexane (v/v), with 0.1% H2SO4. The fatty acid-species composition of this NEFA-fraction is determined as FAME after derivatization with our GC/MS-method on a BPX column (Shimadzu). Validation of the NEFA-method presented was performed in human plasma samples. The validated method has been used with human plasma, cells and tissues, as well as mammalian body fluids and tissue samples. The newly developed solid-phase-extraction (SPE)–GC–MS method allows the rapid separation of the NEFA-fraction from a neutral lipid extract of plasma samples. As a major advantage compared to G–FID-methods, GC–MS allows the use of stable isotope labeled fatty acid precursors to monitor fatty acid metabolism.
Keywords: Non-esterified fatty acids; Free fatty acids; GC–MS; SPE;
Simultaneous determination of olmesartan and amlodipine in human plasma and urine by ultra performance liquid chromatography tandem mass spectrometry by Wenyuan Qi; Qian Zhao; Ji Jiang; Pei Hu (27-34).
A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed to determine olmesartan and amlodipine levels in human plasma and urine simultaneously. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 447 → 207 and 409 → 238 were used to quantify olmesartan and amlodipine, respectively. This assay method has been fully validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. The linearity of this method was found to be within the concentration range of 0.2–500 ng/mL and 4–5000 ng/mL for olmesartan in human plasma and urine and 0.1–50 ng/mL and 2–1000 ng/mL for amlodipine in human plasma and urine. Only 2 min were needed for an analytical run. This assay was used to support a clinical study where multiple oral doses were administered to healthy Chinese subjects to investigate the pharmacokinetics of olmesartan and amlodipine
Keywords: Olmesartan; Amlodipine; UPLC–MS/MS; Human plasma; Urine; Pharmacokinetics;
A rapid and highly sensitive UPLC–MS/MS method using pre-column derivatization with 2-picolylamine for intravenous and percutaneous pharmacokinetics of valproic acid in rats by Kyung-Mi Joo; Dalwoong Choi; Yang-Hui Park; Chang-Geun Yi; Hye-Jin Jeong; Jun-Cheol Cho; Kyung-Min Lim (35-42).
A rapid, highly sensitive and specific ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) for the detection of valproic acid (VPA) in rat plasma following the topical application was developed and validated. This method was carried out with pre-column derivatization using 2-picolylamine (PA) which reacts with the carboxylic acid group of VPA. The derivatization was completed in 10 min and the resulting PA-VPA derivative enabled the sensitive detection of VPA in selected reaction monitoring (SRM) mode. Sample preparation was done with simple liquid–liquid extraction and chromatographic separation was achieved within 5 min on a C18 column using a gradient elution with the mobile phase of 2 mM ammonium formate containing 0.1% formic acid and methanol. The standard curves were linear over the concentration range of 0.07–200 μg/mL with a correlation coefficient higher than 0.99. The limit of detection (LOD) and the lower limit of quantification (LLOQ) was 0.03 and 0.07 μg/mL, respectively with 100 μL of plasma sample. The intra- and inter-day precisions were measured to be below 10.7% and accuracies were within the range of 94.1–115.9%. The validated method was successfully applied to the pharmacokinetics of VPA in the rat following topical and intravenous applications.
Keywords: Valproic acid; 2-Picolylamine; Derivatization; UPLC–MS/MS; Pharmacokinetics; Topical application;
Liquid chromatography–tandem mass spectrometry determination of baclofen in various biological samples and application to a pharmacokinetic study by Tae Hwan Kim; Soyoung Shin; Jeong Cheol Shin; Jin Ho Choi; Won Sik Seo; Gi-Young Park; Dong Rak Kwon; Sun Dong Yoo; Ah-Ram Lee; Sang Hoon Joo; Byung Sun Min; Won Young Yoo; Beom Soo Shin (43-50).
Baclofen is a structural analogue of γ-aminobutyric acid (GABA) that has been used for the treatment of spasticity since 1977. This study describes a simple and sensitive LC/MS/MS assay for the quantification of baclofen in rat plasma, urine, as well as various tissue samples. The assay utilized a simple protein precipitation and achieved lower limit of quantification (LLOQ) of 0.25 ng/mL for rat plasma and brain samples and 2 ng/mL for rat urine, liver and kidney samples. The assay was validated to demonstrate the specificity, linearity, recovery, LLOQ, accuracy, precision, and stability by using matrix matched quality control samples. There is no endogenous or exogenous peaks interfering with the analytes and matrix effects were minimized by optimized separation condition. The assay was linear over a concentration range of 0.25–500 ng/mL for rat plasma and brain tissue, and 2–5000 ng/mL for rat urine, kidney and liver with correlation coefficients >0.999. The mean intra- and inter-day assay accuracies were 94.6–104.6 and 96.0–103.6%, respectively. The mean intra- and inter-day precisions were 5.71 and 5.70%, respectively. The developed assay was successfully applied to a pharmacokinetic study and examined urinary excretion and tissue distribution of baclofen in rats following intravenous and oral administration.
Keywords: Baclofen; LC/MS/MS; Pharmacokinetics; Tissue distribution;
Novel LC–MS/MS method for analyzing imperialine in rat plasma: Development, validation, and application to pharmacokinetics by Qing Lin; Quan Zhang; Xu Song; Tao Gong; Xun Sun; Zhirong Zhang (51-59).
A sensitive, selective, rapid liquid chromatography–electrospray ionization-tandem mass spectrometric method was developed and validated in rat plasma to quantify imperialine, a major active constituent extracted from Bulbus Fritillariae Cirrhosae. Before analysis, plasma samples were pre-treated using cost-effective protein precipitation in order to extract imperialine and the internal standard, carbamzepine. The two analytes were then separated on a Diamonsil ODS chromatography column using gradient elution with a mobile phase of 0.1% aqueous formic acid and acetonitrile. Mass spectrometry was carried out in multiple reaction monitoring mode using a positive electrospray ionization interface. The calibration curve was linear (r 2 = 0.9998) over the concentration range 2–1000 ng/mL, while the validated limit of determination (LOD) was 0.5 ng/mL. Precision varied from 0.1% to 7.1%, and the accuracy varied within ±2%. The method proved robust to sample freezing and thawing, as well as short- and long-term sample storage. The developed method was successfully applied to the pharmacokinetic study of imperialine in rats. Different amounts of imperialine were administered in single doses orally or through the caudal vena cava, and pharmacokinetic parameters were evaluated. Oral bioavailability with a dose of 1 mg/kg was 31.2%; 5 mg/kg, 53.6%; and 10 mg/kg, 47.4%.
Keywords: Imperialine; LC–MS/MS; Validation; Pharmacokinetic study; Rat plasma;
Light scattering coupled with reversed phase chromatography to study protein self-association under separating conditions by Mads Onsberg; Lars H. Øgendal; Marianne L. Jensen; Lotte B. Howells; Birgitte Andersen; Morten J. Bjerrum (60-64).
An on-line method, coupling reversed phase chromatography with static light scattering, was developed to determine the association state of freshly eluted proteins. Under downstream process conditions, human insulin desB30 and human insulin AspB28 were tested at concentrations up to 8.5 mg/mL. The refractive index increment (dn/dc) for insulin was found to depend strongly on the solvent used. A refractive index increment of 0.184 ± 0.003 mL/g was found in an aqueous buffer, pH 7.4, whereas the value was 0.155 ± 0.003 mL/g in 30%, w/w ethanol. The methodology combines on-line SLS and UV measurements with the pre-determined refractive index increment values. The developed on-line method was verified by standard off-line measurements establishing the association state at concentrations between 0.2 and 6.0 mg/mL. The equipment was calibrated utilizing insulin under conditions reported to ensure either monomer or hexamer forms. The self-association of human insulin desB30 was found to be strongly suppressed in 30%, w/w ethanol at pH 7.4 in which the monomer predominates. When stabilized by zinc ions in 30%, w/w ethanol at pH 7.4, an average association number of 3.7 was found. These data demonstrate the effect of ethanol to lower strongly the energy advantage by protein self-association. Potassium chloride and/or calcium chloride in the eluents were found to be of no consequence to the association state.
Keywords: Static light scattering; Equilibrium studies; Zinc; Insulin; Self-association; Hydro-organic solvents;
Ultra-high performance liquid chromatography coupled to mass spectrometry applied to the identification of valuable phenolic compounds from Eucalyptus wood by Sónia A.O. Santos; Carla Vilela; Carmen S.R. Freire; Carlos Pascoal Neto; Armando J.D. Silvestre (65-74).
Ultra-high performance liquid chromatography (UHPLC) was applied for the first time in the analysis of wood extracts. The potential of this technique coupled to ion trap mass spectrometry in the rapid and effective detection and identification of bioactive components in complex vegetal samples was demonstrated. Several dozens of compounds were detected in less than 30 min of analysis time, corresponding to more than 3-fold reduction in time, when compared to conventional HPLC analysis of similar extracts. The phenolic chemical composition of Eucalyptus grandis, Eucalyptus urograndis (E. grandis × E. urophylla) and Eucalyptus maidenii wood extracts was assessed for the first time, with the identification of 51 phenolic compounds in the three wood extracts. Twenty of these compounds are reported for the first time as Eucalyptus genus components. Ellagic acid and ellagic acid-pentoside are the major components in all extracts, followed by gallic and quinic acids in E. grandis and E. urograndis and ellagic acid-pentoside isomer, isorhamnetin-hexoside and gallic acid in E. maidenii. The antioxidant scavenging activity of the extracts was evaluated, with E. grandis wood extract showing the lowest IC50 value. Moreover, the antioxidant activity of these extracts was higher than that of the commercial antioxidant BHT and of those of the corresponding bark extracts. These results, together with the phenolic content values, open good perspectives for the exploitation of these renewable resources as a source of valuable phenolic compounds.
Keywords: UHPLC; Eucalyptus grandis; Eucalyptus urograndis; Eucalyptus maidenii; Phenolic compounds; Wood extracts;
Preparative separation and purification of four cis–trans isomers of coumaroylspermidine analogs from safflower by high-speed counter-current chromatography by Wen-Cong Li; Xiao-Yan Wang; Peng-Cheng Lin; Na Hu; Qiu-Long Zhang; You-Rui Suo; Chen-Xu Ding (75-79).
High-speed counter-current chromatography (HSCCC) was successfully applied for the first time to isolate and purify four cis–trans isomers of coumaroylspermidine analogs from Safflower. HSCCC separation was achieved with a two-phase solvent system composed of chloroform–methanol–water (1:1:1, v/v/v) with the upper phase as the mobile phase. In a single run, a total of 1.3 mg of N 1, N 5, N 10-(E)-tri-p-coumaroylspermidine (EEE), 4.4 mg of N 1(E)-N 5-(Z)-N 10-(E)-tri-p-coumaroylspermidine (EZE), 7.2 mg of N 1(Z)-N 5-(Z)-N 10-(E)-tri-p-coumaroylspermidine (ZZE), and 11.5 mg of N 1,N 5,N 10-(Z)-tri-p-coumaroylspermidine (ZZZ) were obtained from 100 mg of crude sample. High Performance Liquid Chromatography (HPLC) analysis showed that the purities of these four components are 95.5%, 98.1%, 97.5% and 96.2%, respectively. The chemical structures were identified by ESI-MS, 1H NMR and 13C NMR.
Keywords: Safflower; Tri-p-coumaroylspermidine; HSCCC;
Measurement of phenolic environmental estrogens in human urine samples by HPLC–MS/MS and primary discussion the possible linkage with uterine leiomyoma by Fangqing Zhou; Lin Zhang; Ai Liu; Yang Shen; Jinpeng Yuan; Xiaojin Yu; Xu Feng; Qian Xu; Chuange Cheng (80-85).
A method was established for the determination of three phenolic environmental estrogens, namely bisphenol A (BPA), nonylphenol (NP) and octylphenol (OP), in urine from women of uterine leiomyoma group (n = 49) and control group (n = 29), by using solid-phase extraction (SPE) coupled with liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Urine samples were spiked with 2,4,6-tribromophenyl-terminated tetrabromobisphenol-A carbonate oligomer (TBBPA) and nonylphenol D8 (NP-D8) as internal standard (I.S.) and de-conjugated by adding β-glucuronidase and sulfatase before the SPE. The extraction recoveries of BPA, NP and OP were more than 73.3%; the standard curve was linear over the validated concentrations in the range of 1.0–100.0 ng/mL and the limits of detection (LOD) of BPA, NP and OP were 0.32 ng/mL, 0.18 ng/mL and 0.15 ng/mL, respectively. Moreover, by analysing quality control urine samples in 5 days, the results showed that the method was precise and accurate, for the intra- and inter-day CV% within 15.2%. Except that OP was not found (<LOQ) in any of the control urine samples, the three phenolic environmental estrogens were detected in all urine samples. For the uterine leiomyoma women, the mean concentrations of BPA, NP and OP were 13.9 ± 12.7 ng/mL, 2.77 ± 2.22 ng/mL and 4.09 ± 5.51 ng/mL (mean ± SD), respectively. For the control group, the mean concentrations of BPA and NP were 8.50 ± 12.2 ng/mL and 3.84 ± 3.90 ng/mL (mean ± SD), respectively. The Wilcoxon rank sum test was employed for the comparison of BPA and NP between and control in 2 subgroups defined by the number of gravidity (≤3 and >3). NP levels were significantly higher in uterine leiomyoma patients than control group in low gravidity subgroup. Though BPA levels in experimental and control groups were not significantly different, the mean levels and concentration distribution were different. The study suggested that there is certain relationship between exposure concentrations of phenolic environmental estrogens and uterine leiomyoma disease.
Keywords: Bisphenol A; Octylphenol; Nonylphenol; Solid-phase extraction; HPLC–MS; Uterine leiomyoma;
Fast, simultaneous quantification of three novel cardiac drugs in human urine by MEPS–UHPLC–MS/MS for therapeutic drug monitoring by Sylwia Magiera (86-95).
A sensitive and selective ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) method was developed for the fast, simultaneous quantification of three novel cardiac drugs (aliskiren, prasugrel and rivaroxaban) in human urine. Sample preparation was performed with microextraction with packed sorbent (MEPS), which is a miniaturization of solid phase extraction. The optimal conditions for MEPS extraction were obtained using C8 sorbent, small sample volumes and a short time period (about 3 min for the entire sample preparation step). Chromatographic separation of the selected compounds was achieved in less than 1.5 min on a Zorbax Rapid Resolution High Definition SB-C18 column using isocratic elution with 0.1% formic acid and acetonitrile (70:30, v/v) at a flow rate of 0.8 mL min−1. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via an electrospray ionization source with positive ionization mode. The method was fully validated according to the latest recommendations of international guidelines. The lower limit of quantification was 5.0 pg mL−1 for aliskiren and rivaroxaban and 0.5 pg mL−1 for prasugrel. The intra- and inter-day precision was within 7.12% and the accuracy ranged from −7.54% to 4.17%. The mean extraction recoveries of the MEPSC8 methodology were found to be 98.3% for aliskiren, 100.3% for rivaroxaban and 99.9% for prasugrel. This MEPSC8-UHPLC-MS/MS method offers a fast, simple and precise way to determine selected novel cardiac drugs in human urine that could be applied to therapeutic drug monitoring and pharmacokinetic studies.
Keywords: Drugs; Liquid chromatography; Mass spectrometry; Method validation; Microextraction by packed sorbent;
High sensitivity LC–MS/MS method for direct quantification of human parathyroid 1–34 (teriparatide) in human plasma by Erin E. Chambers; Mary E. Lame; Jon Bardsley; Sally Hannam; Cristina Legido-Quigley; Norman Smith; Kenneth J. Fountain; Eileen Collins; Elizabeth Thomas (96-104).
Teriparatide, the 1–34 fragment of human parathyroid hormone, is used to treat osteoporosis patients with a high risk of fracture by stimulating new bone formation. Routinely teriparatide is quantified using radioimmunoassay however the LC–MS/MS described here has the potential to achieve greater accuracy and precision, higher specificity, and is readily implemented in routine bioanalytical laboratories. Hence a complete method combining effective sample prep with appropriate LC separation and selected reaction monitoring (SRM) MS detection was developed to selectively separate teriparatide from closely related endogenous peptides and to reduce interferences. Samples were concentrated without evaporation, minimizing the risk of adsorptive losses. Chromatography was performed on a sub 2 μm particle charged surface hybrid column, which provided significantly higher peak capacity than a traditional C18 column when formic acid was used as the mobile phase modifier. Total LC cycle time was 6 min. An LOD of 15 pg/mL (3.6 fmol/mL) from 200 μL of human plasma was readily achieved and standard curves were accurate and precise from 15 pg/mL to 500 pg/mL. Mean QC accuracies ranged from 90% to 106%. Mean QC precision was better than 7%. The CV of matrix factors across 6 sources of human plasma was 5%. The assay presented here is the first LC–MS method which reaches clinically relevant detection limits for teriparatide
Keywords: Human growth hormone; rhPTH (1–34); Bioanalysis; LC–MS/MS peptide quantification; Solid phase extraction (SPE); Teriparatide;
The application of an LC-MS/MS method in a pharmacokinetic study for the determination of the concentration of nifekalant in human plasma by Mengmeng Zhang; Guobing Shi; Yin Sui; Ye An; Ming Yan; Yunbiao Tang (105-110).
In the present study, a simple, rapid, and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the concentration of Nifekalant in human plasma was developed and validated. The analyte and the internal standard were extracted from human plasma using dichloromethane and analyzed using an ultra-fast liquid chromatographer (UFLC) coupled to an electrospray ionization (ESI) tandem mass spectrometer in the positive mode. The chromatographic analysis was performed isocratically on an Inertsil ODS-SP column (150 mm × 4.6 mm I.D., 5 μm). The mobile phase was a mixture of 15% 10 mM aqueous ammonium formate and 85% methanol (the pH was adjusted to 3.5 with formic acid) at a flow rate of 0.8 mL/min with a split ratio of 1:1 to the ionization source. The lower limit of quantification (LLOQ) was 5.05 ng/mL, and a linear calibration curve was obtained over the concentration range of 5.05 to 3030 ng/mL. The intra-day and inter-day assay variations were less than 9.06%, and the accuracy values (relative error) were in the range of −10.95% to 2.27%. The essential pharmacokinetic parameters of the intravenously injection of Nifekalant were found to be the following: t 1/2 = 1.26 ± 0.16 h, C max = 1.943 ± 0.411 mg/L, and AUC0–12h = 4.600 ± 0.756 mg/L·h.
Keywords: Nifekalant; Pharmacokinetics; LC-MS/MS;
Anion-exchange purification of recombinant factor IX from cell culture supernatant using different chromatography supports by Daniel A. Ribeiro; Douglas F. Passos; Helen C. Ferraz; Leda R. Castilho (111-118).
Both recombinant and plasma-derived factor IX concentrates are used in replacement therapies for the treatment of haemophilia B. In the present work, the capture step for a recombinant FIX (rFIX) purification process was investigated. Different strong anion-exchange chromatography media (the resins Q Sepharose® FF and Fractogel® TMAE, the monolith CIM® QA and the membrane adsorber Sartobind® Q) were tested for their rFIX binding capacity under dynamic conditions. In these experiments, crude supernatant from CHO cells was used, thus in the presence of supernatant contaminants and mimicking process conditions. The highest dynamic binding capacity was obtained for the monolith, which was then further investigated. To study pseudoaffinity elution of functional rFIX with Ca2+ ions, a design of experiments to evaluate the effects of pH, NaCl and CaCl2 on yield and purification factor was carried out. The effect of pH was not statistically significant, and a combination of no NaCl and 45 mM CaCl2 yielded a good purification factor combined with a high yield of active rFIX. Under these conditions, activity yield of rFIX was higher than the mass yield, confirming selective elution of functional, γ-carboxylated rFIX. Scaling-up of this process 8 fold resulted in very similar process performance. Monitoring of the undesired activated FIX (FIXa) revealed that the FIXa/FIX ratio (1.94%) was higher in the eluate than in the loaded sample, but was still within an acceptable range. HCP and DNA clearances were high (1256 and 7182 fold, respectively), indicating that the proposed process is adequate for the intended rFIX capture step.
Keywords: Anion exchange chromatography; Recombinant factor IX (rFIX); Activated factor IX (FIXa); Resins, monoliths and membrane adsorbers; Pseudoaffinity elution with calcium chloride; Host-cell protein and residual DNA clearance;