Rational methods for predicting human monoclonal antibodies retention in protein A affinity chromatography and cation exchange chromatography. Structure-based chromatography design for monoclonal antibodies

Rational methods for predicting the chromatographic behavior of human monoclonal antibodies (hMabs) in protein A affinity chromatography and cation exchange chromatography from the amino acid sequences information were proposed. We investigated the relation between the structures of 28 hMabs and their chromatographic behavior in protein A affinity chromatography and cation exchange chromatography using linear gradient elution experiments. In protein A affinity chromatography, the elution pH of the hMabs was correlated with not only the structure of the Fc region (subclass), but also that of the variable region. The elution pH of hMabs that have LYLQMNSL sequences in between the CDR2 and CDR3 regions of the heavy chain became lower among the same subclass of hMabs. In cation exchange chromatography, the peak salt concentrations IR of hMabs that have the same sequences of variable regions (or that have a structural difference in their Fc region, which puts them into a subclass) were similar. The IR values of hMabs were well correlated with the equilibrium association constant Ke, and also with the surface positive charge distribution of the variable region of the heavy chain (corrected surface net positive charge (cN) of the VH region). Based on these findings, we developed rational methods for predicting the retention behavior, which were also tested with eight additional hMabs. By considering the information on the number of binding sites associated with protein adsorption as determined experimentally, and the surface positive charge distribution from the three-dimensional structure of Mab A, we hypothesized that hMabs is separated by cation exchange chromatography as the surface positive charge distribution of the VH region is recognized.     

Analytical and preparative separation of PEGylated lysozyme for the characterization of chromatography media

The effect of PEGylation on cation exchange chromatography was studied with poly(ethylene glycol) of different chain lengths (5 kDa, 10 kDa and 30 kDa) using lysozyme as a model system. A stable binding via reduction of a Schiff base was formed during random PEGylation on lysine residues with methoxy-PEG-aldehyde. A purification method for PEGylated proteins using cation exchange chromatography was developed, and different isoforms of mono-PEGylated lysozyme were isolated. TSKgel SP-5PW and Toyopearl GigaCap S-650M showed the best performance of all tested cation exchange resins, and the separation of PEGylated lysozyme could be also scaled up to semi-preparative level. Size-exclusion chromatography, SDS-PAGE and MALDI-TOF mass spectrometry were used for analysis. Separated mono-PEGylated lysozyme of different sizes was used to determine dynamic binding capacities (DBC) and selectivity of cation exchange chromatography resins. An optimization of binding conditions resulted in a more than 20-fold increase of DBC for Toyopearl GigaCap S-650M with 30 kDa mono-PEGylated lysozyme.     

Isolation and desalting with cation‐exchange chromatography for compound‐specific nitrogen isotope analysis of amino acids: application to biogeochemical samples

We have established a procedure for removing interfering materials from extracts of geological and biological samples, in order to determine precise compound‐specific nitrogen isotopic compositions of amino acids. We employed cation‐exchange chromatography of protein and non‐protein amino acids prior to derivatization for gas chromatographic separation. The average recovery of a standard amino acid solution was better than 94%, without nitrogen isotope fractionation during the cation‐exchange chromatography. We applied the procedure to various environmental samples including ‘hard’ (calcareous, siliceous, rock and sediment samples) and ‘soft’ materials (aggregated microbial samples and biological soft tissue samples). We conclude that cation‐exchange chromatography is a pre‐treatment procedure which should be widely useful for the determination of compound‐specific nitrogen isotopic compositions of amino acids. Copyright © 2010 John Wiley & Sons, Ltd.     

Preparation of weak cation exchange packings for chromatographic separation of proteins using "click chemistry'

"Click chemistry" is defined as a class of robust and selective chemical reactions affording high yields and is tolerant to a variety of solvents (including water), functional groups, and air. In this study, click chemistry was used as an effective strategy for coupling three alkyne-carboxylic acids onto the azide-silica to obtain three novel stationary phases of weak cation exchange chromatography, which were characterized with FTIR and elemental analysis. Six kinds of standard proteins, such as myoglobin, RNase A, RNase B, cytochrome C, α-chymotrypsin A, and lysozyme, were separated completely with the three novel weak cation exchange chromatography stationary phases. Compared with commercial weak cation exchange chromatography columns, the three kinds of novel weak cation exchange chromatography packings prepared by click chemistry approach have better resolution and selectivity. The mass recovery of more than 97% was obtained for all the tested proteins, and the bioactivity recovery of lysozyme on the prepared column was determined to be 96%. In addition, lysozyme was purified successfully from egg white with the novel weak cation exchange chromatography column by one step. The purity was more than 97% and a high specific activity was achieved to be 81 435 U/mg. The results illustrate the potential of click chemistry for preparing stationary phase for ion-exchange chromatography.     

高效阳离子交换法分离纯化蛋清中的溶菌酶

 建立了用高效阳离子交换分离纯化蛋清中溶菌酶的新方法 ,讨论了纯化的工艺条件。蛋清样品匀浆后 ,用氯化钠初步纯化 ,然后用弱阳离子交换柱XIDACE WCX分离。结果表明 ,被纯化的溶菌酶和杂蛋白得到很好分离。经活性检测 ,溶菌酶过柱后的活性回收率为 10 7% ,蛋白的比活为 15 4 6 7U/mg ,纯化倍数提高了 5 6倍。用尺寸排阻 (SEC)鉴定 ,得到的溶菌酶呈均一性。该法较传统软基质低压离子交换分离速度快 ,纯化效率高。     

Application of arginine as an efficient eluent in cation exchange chromatographic purification of a PEGylated peptide

A cation exchange chromatographic purification process step was developed for the purification of human PEGylated PYY 3–36 from the PEGylation reaction mixture. In this publication we describe experiments carried out to evaluate the chromatographic performance of arginine chloride as an effective cation exchange chromatography eluent. Using arginine we obtained improved recovery and resolution during chromatographic purification of a peptide PEGylation reaction mixture. The chromatographic elution performance of arginine was compared to other cationic amino acids and sodium chloride. Arginine provided higher yield and better resolution of product from other process impurities. The process was successfully scaled up to produce clinical supplies. The basis for improvement in process performance with arginine was characterized by examining the effect of buffer and concentration of the PEGylated peptide on hydrodynamic volume of the molecule in solution. These results were used to predict the behavior of the molecule in the chromatography process. The enhanced chromatographic performance could be attributed to changes in molecular size with concentration, higher eluent strength of arginine, and resulting changes in mass transfer resistance.     

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification

Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb's commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes.     

Development and Validation of Salt Gradient CEX Chromatography Method for Charge Variants Separation and Quantitative Analysis of the IgG mAb-Cetuximab

Ion exchange chromatography is widely used for charge variant analysis of proteins, including monoclonal antibodies. In this study, a simple and robust salt gradient cation exchange chromatography was developed and validated for quantitative determination of cetuximab in biopharmaceutical formulations. For this purpose, we investigated the effect of various parameters including buffer composition, column temperature, pH, gradient volume and flow rate on chromatographic separation of charge variants to achieve the acceptable peak separation, and the optimum condition was selected. Validation of the method was done in accordance with the International Conference on Harmonization (ICH) guidelines. The developed method was found to provide a linear regression over the concentration range of 0.06–2.00 mg mL^?1 yielding a correlation coefficient of 0.9972. The limits of detection and quantification for the developed method were 0.02 and 0.06 mg mL^?1, respectively. The intra-day and inter-day precision had relative standard deviation values?≤?2.7%. The robustness of the method was assessed by changes in the applied pH range of buffer, temperature, mobile phase composition, and flow rate. Specificity of the method was confirmed by evaluation of baseline resolution of the mAb variants from product excipients, which showed no interference between excipients and cetuximab. The stability indicating capability of this method was determined using photodegraded, and mechanically and thermally stressed samples. The proposed method could be applied as a simple, precise, and robust quantitative technique which can be reproduced in any labs for the high-throughput quality control and stability assessment of in-process and final product samples.     

Selective retention of basic compounds by metal aquo‐ion affinity chromatography

A novel metal aquo‐ion affinity chromatography has been developed for the analysis of basic compounds using heat‐treated silica gel containing hydrated metal cations (metal aquo‐ions) as the packing material. The packing materials of the metal aquo‐ion affinity chromatography were prepared by the immobilization of a single metal component such as Fe(III), Al(III), Ag(I), and Ni(II) on silica gel followed by extensive heat treatment. The immobilized metals form aquo‐ions to present cation‐exchange ability for basic analytes and the cation‐exchange ability for basic analytes depends on pK_a of the immobilized metal species. In the present study, to evaluate the retention characteristics of metal aquo‐ion affinity chromatography, the on‐line solid‐phase extraction of drugs was investigated. Obtained data clearly evidence the selective retention capability of metal aquo‐ion affinity chromatography for basic analytes with sufficient capacity.     

Fully automated micro- and nanoscale one- or two-dimensional high-performance liquid chromatography system for liquid chromatography-mass spectrometry compatible with non-volatile salts for ion exchange chromatography

A one- or two-dimensional high performance liquid chromatography system for electrospray ionization mass spectrometers has been developed that is optimized for ion exchange and reversed phase separations. A unique and simple valve configuration permits the use of a variety of non-volatile salts; ammonium sulfate was used in an example of strong cation exchange separations. The system was designed and evaluated for both micro- and nanoflow chromatography. The peptide detection limit was approximately 100 fmol for micro- and 20 fmol for nanoflow, demonstrating the concentration and mass sensitivity improvements expected with nanoelectrospray ionization. The 1D/2D-HPLC MS system is fully automated for routine peptide analyses, compatible with direct injection of proteolytic digests, and exhibits chromatographic reproducibility and sensitivity. Software permits operator selection of either a 1D or 2D configuration with corresponding system parameters as required for individual samples. The hardware elements and resulting performance are described in this paper.     

Weak anion and cation exchange mixed-bed microcolumn for protein separation

To separate proteins with a wide distribution of pIs under the conditions compatible to online tryptic digestion (with preferable pH=8.0), weak anion and cation exchange chromatography (WAX/WCX) mixed‐bed microcolumn has been developed. With a mixture of five proteins with pIs ranging from 4.2 to 11.4, the effect of WAX/WCX ratio on the separation performance was investigated, and an optimum packing ratio of 1:1 w/w was obtained. Moreover, the undesirable hydrophobic interaction between the proteins and the stationary phase was suppressed with 10% ACN v/v added in the mobile phases. Under the optimized conditions compatible to tryptic digestion, basic and acidic proteins were resolved simultaneously, with RSDs of relative retention time on six columns less than 6%, indicating the good resolution and packing reproducibility. Furthermore, one RPLC fraction of proteins extracted from rat middle brain and the whole protein mixture extracted from rat liver were analyzed, respectively. The results demonstrated better separation performance on WAX/WCX microcolumns than that on both weak anion exchange chromatography and weak cation exchange chromatography at pH ～8. We anticipate that WAX/WCX microcolumns are promising for the integration of protein separation and tryptic digestion aiming at high‐throughput proteome study.     

Chromatographic purification of human α1 proteinase inhibitor from dissolved Cohn fraction IV-1 paste

A novel chromatographic process for purification of α₁ proteinase inhibitor (α₁-PI) from Cohn fraction IV-1 paste is described. This process has been successfully scaled up to 50-l columns. It involves DEAE chromatography, sulfopropyl (S) cation chromatography, tri-n-butyl phosphate (TNBP)–cholate treatment, a second S cation chromatography, freeze–drying and dry-heat. The process has been optimized for purity, yield, lipid removal, chemical usage and water consumption. Filtration after TNBP–cholate treatment plays a key role in ensuring a low lipid content in the final product. Pre-equilibration with high salt buffer is necessary to reduce the water consumption significantly during the ion-exchange chromatography equilibration step. The final product is approximately 95% pure by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, with a 64% to 70% yield from IV-1 paste.     

A new approach to ion exchange chromatography with conductivity detection for adulterants investigation in dietary supplements

The extent of adulteration of dietary supplements has significantly increased in recent years. This situation worries health authorities and requires auxiliary analytical tools for the investigation of illegal substances purposely added. Ion exchange chromatography with conductivity detection is a consolidated analytical technique for the determination of inorganic compounds in various matrices. This technique has been applied to the pharmaceutical characterization of mainly impurities and degradation products. This work presents a new approach to ion exchange chromatography as a screening method to investigate the presence of amfepramone, femproporex, sibutramine, bisacodyl and amiloride in dietary supplements advertised for weight loss. The method was optimized and validated using a Metrosep C4 100/4.0 cation exchange column. The mobile phase consisted of 1.8 mm HNO₃ containing 2% acetonitrile (v/v), with a flow rate of 0.9 ml min^?1, and nonsuppressed conductivity detection was applied. The limits of detection and quantification varied from 1.01 to 3.62 mg L^?1 and from 1.48 to 8.72 mg L^?1, respectively. The proposed method was successful applied to 78 solid dietary supplement samples, in two of which adulterations were found. Moreover, ion exchange chromatography with conductivity detection could be easily used for quality control without prior complex sample pre‐treatment.     

Determination of triethylenetetramine in plasma of patients by high-performance liquid chromatography

A sensitive and simple fluorometric method for the determination of N,N'-bis(2-aminoethyl)-1,2-ethanediamine dihydrochloride (triethylenetetramine) in human plasma by high-performance liquid chromatography is described. Free triethylenetetramine (TETA) obtained by passing the TETA-copper chelate compound through a solid-phase cation exchange resin was converted to its fluorescamine derivative in the presence of ethylenediaminetetraacetic acid to mask the interfering metal ions in the reaction solution, and the derivatives were separated on a nitrile high-performance liquid chromatograph column (Nucleosil 5-CN) using isocratic elution. The plasma levels of TETA were measured in eight patients receiving treatment for excess copper. Absorption rates of TETA were relatively slow and the peak levels were significantly different among patients. The bioavailability of TETA in the rat was also examined and the ratio of intestinal absorption was extremely low.     

Insulins in equine urine: qualitative analysis by immunoaffinity purification and liquid chromatography/tandem mass spectrometry for doping control purposes in horse-racing

Insulin is a peptide hormone consisting of two peptide chains (A- and B-chain) that are cross-linked by two disulfide bonds. To obtain improved pharmacokinetic onset of action profiles of insulin treatment in diabetic patients, recombinant long-, intermediate-, and rapid-acting insulin analogs are produced, in which the C-terminal end of the B-chain plays an especially important role.A review of the veterinary literature reveals the low prevalence of equine type I diabetes mellitus, which indicates that the therapeutic use of insulin in racing horses is unlikely. Although there is no unequivocal evidence of an overall performance-enhancing effect of insulin, in human sports the misuse of insulin preparations is reported among elite athletes. The desired effects of insulin include the increase of muscular glycogen prior to sports event or during the recovery phase, in addition to a chalonic action, which increases the muscle size by inhibiting protein breakdown.In the present study urinary insulin was detected in equine samples and differences between equine insulin, human insulin, as well as rapidly acting recombinant insulin variants were examined. The method was based on sample purification by solid-phase extraction (SPE) and immunoaffinity chromatography (IAC), and subsequent analysis by microbore liquid chromatography (LC) and tandem mass spectrometry (MS/MS) using top-down sequencing for the determination of various insulins. Product ion scan experiments of intact proteins and B-chains enabled the differentiation between endogenously produced equine insulin, its DesB30 metabolite, human insulin and recombinant insulin analogs, and the assay allowed the assignment of individual product ions, especially those originating from modified C-termini of B-chains.     

双柱吸附法对莲子心总碱的分离纯化研究

研究了大孔吸附树脂和阳离子交换树脂双柱吸附法对莲心碱、异莲心碱和甲基莲心碱的吸附行为。利用阳离子交换树脂D72制备出莲心碱、异莲心碱和甲基莲心碱,含量分别为10.6%、10.6%和28.0%,总含量为49.2%的莲子心总碱提取物,其中杂质主要是水溶性较大的生物碱成分。利用AKS-W大孔吸附树脂能去除极性较大的生物碱,而D72阳离子交换树脂能去除非生物碱的特点,采用双柱联合吸附法,制备出3种吸附质含量分别为33.1%、15.0%和34.5%,总含量为82.6%的莲子心总碱提取物。     

ISOLATION AND PURIFICATION OF ALKALOIDS FROM PLUMULA NELUMBINIS BY DOUBLE-COLUMN ADSORPTION CHROMOTOGRAPHY

The performance of adsorption and separation for liensinine, isoliensinine and neferine was studied by double-column adsorption chromatography using macroporous adsorption and cation exchange resins. The alkaloid extract with 49.2% total contents by mass representing 10.6% liensinine, 10.6% isoliensinine and 28.0% neferine respectively was prepared by D72 cation exchange resins, in which most of the impurities were water-soluble alkaloids. Furthermore, the alkaloid extract with 82.6% total contents by mass containing 33.1%, 15.0% and 34.5% of the three adsorbates respectively was prepared by double-column adsorption chromatography using AKS-W macroporous adsorption and D72 cation exchange resins. As a result, the content of single and total alkaloids has been greatly increased by the double-column adsorption chromatography.     

姜黄素前药的研究进展

近年来,天然产物姜黄素因其显著的抗肿瘤等多种生物活性引起广泛关注.然而姜黄素水溶性差、化学结构不稳定、生物利用度低,限制了其在临床上的进一步应用.用小分子或大分子载体对姜黄素的酚羟基进行修饰制备得到的姜黄素前药能较好地解决姜黄素上述的缺点,也是改善姜黄素成药性的有效策略.综述了近年来国内外姜黄素前药的研究进展.     

Determination of isoflavones in rat serum using liquid chromatography–tandem mass spectrometry with a highly efficient core–shell column

Consumption and nutritional supplementation of soy and soy-based products have been linked to health benefits such as lower cholesterol and triglyceride levels, and decreased incidence of cardiovascular disease and diabetes. In this study, we have developed a sensitive, specific, and robust method using high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) for determination of serum isoflavones. A new highly efficient pentafluorophenyl phase core–shell column was first used to separate all isoflavones within 3 min, a separation time which is comparable to ultra-pressure liquid chromatography (UPLC) and micro-HPLC. A two-enzyme hydrolysis system with sulfatase and β-glucuronidase has also been developed to improve the efficiency of deconjugation of conjugated isoflavones in serum. The corresponding conjugated isoflavones were used to evaluate recoveries. In addition to duplicates, the method of standard addition was also applied in sample analysis for quality control. The developed method was applied to the analysis of 32 serum samples and was shown to be specific, sensitive and reproducible.     

Chromatographic Processing Scheme for Continuous Harvesting of rec-Prourokinase from Serum Free Medium Cell Cultures

Continuous perfusion cell culture, using albumin containing medium, offers the potential advantages of higher recombinant Prourokinase (r-ProUK) yields, higher initial product purity and increased throughput compared to batch culture technology using medium supplemented with fetal bovine serum. We have characterized the production of r-ProUK in medium supplemented with a lipid rich bovine serum albumin (Albumax) in a perfusion system. The results of these studies showed that it was necessary to modify the r-ProUK batch recovery scheme to process r-ProUK from a perfusion system. To accommodate large volumes of perfusate harvested over a ten to fourteen day production cycle, cation exchange and hydrophobic interaction chromatography (HIC) resins were identified that had increased product binding capacity, better flow characteristics and wider pH ranges which allowed caustic cleaning. The mobile phase composition, pH and ionic strength were modified to improve r-ProUK yields from the identified resins, and procedures were developed to eliminate r-ProUK degradation products. Strategies were defined for processing continuous harvest, which contained four to seven times the amount of r-ProUK of batch harvests.