Direct coupling of size exclusion chromatography and mass spectrometry for the characterization of complex monoclonal antibody products

The present study describes the possibilities offered by an innovative bioinert size exclusion chromatography column for size variant characterization of complex monoclonal antibody products. This size exclusion chromatography column includes a novel column hardware surface. The column was prepared from metallic hardware components that were treated to have prototype hydrophilically modified hybrid organic–inorganic silica surfaces called hybrid surface technology. This provides a significant reduction in nondesired hydrophobic and electrostatic interactions that can occur between column and analyte when performing size exclusion chromatography analysis with volatile mobile phase. Compared to a reference stainless-steel column packed with the same batch of packing material, peak tailing, band broadening, and above all recovery of high molecular weight species were distinctly improved for all types of monoclonal antibody products. Based on our observations, we found that 50 mM ammonium acetate in water was a suitable mobile phase offering good compromise in terms of liquid chromatography performance and mass spectrometry sensitivity. In addition, method repeatability (intra- and interday relative standard deviations) on elution times and high molecular weight species peak areas were found to be excellent. By using this innovative size exclusion chromatography material, the low and high molecular weight species contained in various stressed and nonstressed monoclonal antibody products were successfully characterized with mass spectrometry detection.     

Application of a novel affinity adsorbent for the capture and purification of recombinant Factor VIII compounds

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13 kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.     

Accelerated purification process development of monoclonal antibodies for shortening time to clinic. Design and case study of chromatography processes

For accelerating the purification process development of human monoclonal antibodies (hmAbs) for pharmaceutical drugs, we designed a standardized method for setting the conditions of the purification process, which could be applied to hmAbs for the early phase of pharmaceutical development. The process includes three sequential chromatography steps: Protein A affinity chromatography (AFC), anion-exchange chromatography (AIEC) and cation-exchange chromatography (CIEC), and also includes a low pH virus inactivation step after the AFC step. We predicted the elution pH in the AFC and elution salt concentration in the CIEC from the amino acid sequences of hmAbs, as described in our previous paper. The mobile phase pH in AIEC and the pH for virus inactivation were also predicted based on the amino acid sequence of hmAb. As a case study, six hmAbs (two of IgG(1), two of IgG(2) and two of IgG(4)) were purified with the standardized method. The recovery, purity and clearance of impurities (DNA, host cell proteins (HCP), and Protein A) were examined. All the six hmAbs were purified with high recovery and high clearance of the impurities. Factors affecting the impurities level in the purified products are also discussed.     

Development and application of immunoaffinity column chromatography for atrazine in complex sample media

A rabbit antibody immunoaffinity (IA) column procedure was evaluated as a cleanup method for the determination of atrazine in soil, sediment, and food. Four IA columns were prepared by immobilizing a polyclonal rabbit anti-atrazine antibody solution to HiTrap Sepharose columns. Atrazine was bound to the IA columns when the loading solvents were either 100% water, 2% acetonitrile in water, or 10% methanol in phosphate buffered saline (PBS). Quantitative removal of atrazine from the IA columns was achieved with elution solvents of either 70% ethanol in water, 70% methanol in water, or 100% methanol. One control column was prepared using nonspecific rabbit IgG antibody. This control column did not retain any applied atrazine indicating atrazine did not bind indiscriminately to protein or the Sepharose support. The four IA columns showed reproducible coupling efficiency for the immobilization of the atrazine antibody and consistent binding and releasing of atrazine. The coupling efficiency (4.25 mg of antibody in 1 mL of resin bed) for the four IA columns ranged from 93 to 97% with an average of 96 ± 2% (2.1%). Recoveries of the 500, 50, and 5 ng mL⁻¹ atrazine standard solutions from the four IA columns were 107 ± 7% (6.5%), 122 ± 14% (12%), and 114 ± 9% (8.0%) respectively, based on enzyme-linked immunosorbent assay (ELISA) data. The maximum loading was approximately 700 ng of atrazine for each IA column (∼0.16 μg of atrazine per mg of antibody). The IA columns could withstand 100% methanol as the elution solvent and could be reused more than 50 times with no change in performance. The IA columns were challenged with soil, sediment, and duplicate-diet food samples and effectively removed interferences from these various matrices for subsequent gas chromatography/mass spectrometry (GC/MS) or ELISA analysis. The log-transformed ELISA and GC/MS data were significantly correlated for soil, sediment and food samples although the ELISA values were slightly higher than those obtained by GC/MS. The IA column cleanup procedure coupled with ELISA analysis could be used as an alternative effective analytical method for the determination of atrazine in complex sample media such as soil, sediment, and food samples.     

Automated flow-based anion-exchange method for high-throughput isolation and real-time monitoring of RuBisCO in plant extracts

In this work, a miniaturized, completely enclosed multisyringe-flow system is proposed for high-throughput purification of RuBisCO from Triticum aestivum extracts. The automated method capitalizes on the uptake of the target protein at 4 °C onto Q-Sepharose Fast Flow strong anion-exchanger packed in a cylindrical microcolumn (105 × 4 mm) followed by a stepwise ionic-strength gradient elution (0-0.8 mol/L NaCl) to eliminate concomitant extract components and retrieve highly purified RuBisCO. The manifold is furnished downstream with a flow-through diode-array UV/vis spectrophotometer for real-time monitoring of the column effluent at the protein-specific wavelength of 280 nm to detect the elution of RuBisCO. Quantitation of RuBisCO and total soluble proteins in the eluate fractions were undertaken using polyacrylamide gel electrophoresis (PAGE) and the spectrophotometric Bradford assay, respectively. A comprehensive investigation of the effect of distinct concentration gradients on the isolation of RuBisCO and experimental conditions (namely, type of resin, column dimensions and mobile-phase flow rate) upon column capacity and analyte breakthrough was effected. The assembled set-up was aimed to critically ascertain the efficiency of preliminary batchwise pre-treatments of crude plant extracts (viz., polyethylenglycol (PEG) precipitation, ammonium sulphate precipitation and sucrose gradient centrifugation) in terms of RuBisCO purification and absolute recovery prior to automated anion-exchange column separation. Under the optimum physical and chemical conditions, the flow-through column system is able to admit crude plant extracts and gives rise to RuBisCO purification yields better than 75%, which might be increased up to 96 ± 9% with a prior PEG fractionation followed by sucrose gradient step.     

Application of a chromatography model with linear gradient elution experimental data to the rapid scale-up in ion-exchange process chromatography of proteins

We applied the model described in our previous paper to the rapid scale-up in the ion exchange chromatography of proteins, in which linear flow velocity, column length and gradient slope were changed. We carried out linear gradient elution experiments, and obtained data for the peak salt concentration and peak width. From these data, the plate height (HETP) was calculated as a function of the mobile phase velocity and iso-resolution curve (the separation time and elution volume relationship for the same resolution) was calculated. The scale-up chromatography conditions were determined by the iso-resolution curve. The scale-up of the linear gradient elution from 5 to 100mL and 2.5L column sizes was performed both by the separation of beta-lactoglobulin A and beta-lactoglobulin B with anion-exchange chromatography and by the purification of a recombinant protein with cation-exchange chromatography. Resolution, recovery and purity were examined in order to verify the proposed method.     

Pre-concentration of Cd, Co, Cu, Ni and Zn using different off-line ion exchange procedures followed by the inductively coupled plasma atomic emission spectrometric detection

The chelating resin Metalfix Chelamine and the strong cation exchanger Dowex 50W-X4 were studied for the off-line pre-concentration of Cd, Co, Cu, Ni and Zn prior to the measurements by the inductively coupled plasma atomic emission spectrometry. Different approaches to the recovery of metals were investigated and compared: the elution with the solutions of HCl and HNO₃ and the decomposition of the resins with the adsorbed metals by means of the high-pressure microwave assisted system. Due to the problem arisen with the quantitative elution of the metals from the resins, which bound the analytes very strongly, the procedure of the digestion of the resin after the pre-concentration step was preferable to the plain elution, giving significantly better recoveries of the metals. The chosen procedure was applied for the determination of traces of Cd, Co, Cu, Ni and Zn in beer. For the analysis, mineralised and not mineralised beer samples were treated with Dowex 50W-X4 resin in order to assess the total content of Cd, Co, Cu, Ni and Zn and the fraction of the cationic labile species of the analytes, respectively. The increase in the sensitivity allowed determination of the selected metals at concentrations of order of 1 ng ml⁻¹. The accuracy of the entire procedure was verified by the recovery test in the spiked samples of beer. The proposed method provides full recovery for Cd (101±1%), Co (99±2%) and Zn (106±3%) and reasonably high recovery for Cu (84±1%) and Ni (89±1%).     

Recovery of actinorhodin from fermentation broth

In the present work, a new method of purification for actinorhodin was developed using an expanded bed chromatography technique in which antibiotic capture, feedstock clarification, centrifugation, dialysis and concentration are done in one step. The cation-exchanger (P-11) resulted in 26% adsorption and 2% recovery whereas the anion-exchanger (DE-52) resulted in 99% adsorption and 56% recovery of adsorbed antibiotic using methanol buffer and 2 M NH4Cl as eluting agent. Streamline DEAE anion-exchanger, which is especially designed for EBA applications, yields 82% adsorption and 50% elution of actinorhodin fed into the chromatography column directly from the fermentation broth. Isocratic elution resulted in extremely efficient yield compared to linear gradient elution, i.e. 13.5-fold more recovery in the column with an aspect ratio (L:D) of 4. Expansion by 150% of settled bed resulted in the best recovery of actinorhodin among 100 and 200% expansions. A comparison of breakthrough profiles in packed and expanded bed adsorption showed that the performance of the expanded bed is better (by 33%) at allowing more volume of the fermentation broth to pass through the chromatography column.     

Matrix-assisted refolding of autoprotease fusion proteins on an ion exchange column

Refolding of proteins must be performed under very dilute conditions to overcome the competing aggregation reaction, which has a high reaction order. Refolding on a chromatography column partially prevents formation of the intermediate form prone to aggregation. A chromatographic refolding procedure was developed using an autoprotease fusion protein with the mutant EDDIE from the N^pro autoprotease of pestivirus. Upon refolding, self-cleavage generates a target peptide with an authentic N-terminus. The refolding process was developed using the basic 1.8-kDa peptide sSNEVi-C fused to the autoprotease EDDIE or the acidic peptide pep6His, applying cation and anion exchange chromatography, respectively. Dissolved inclusion bodies were loaded on cation exchange chromatographic resins (Capto S, POROS HS, Fractogel EMD SO₃⁻, UNOsphere S, SP Sepharose FF, CM Sepharose FF, S Ceramic HyperD F, Toyopearl SP-650, and Toyopearl MegaCap II SP-550EC). A conditioning step was introduced in order to reduce the urea concentration prior to the refolding step. Refolding was initiated by applying an elution buffer containing a high concentration of Tris–HCl plus common refolding additives. The actual refolding process occurred concurrently with the elution step and was completed in the collected fraction. With Capto S, POROS HS, and Fractogel SO₃⁻, refolding could be performed at column loadings of 50 mg fusion protein/ml gel, resulting in a final eluate concentration of around 10–15 mg/ml, with refolding and cleavage step yields of around 75%. The overall yield of recovered peptide reached 50%. Similar yields were obtained using the anion exchange system and the pep6His fusion peptide. This chromatographic refolding process allows processing of fusion peptides at a concentration range 10- to 100-fold higher than that observed for common refolding systems.     

Modelling of elution-extrusion counter-current chromatography using perfect replacement approach

Basing on the perfect replacement approach the equilibrium cell model is developed to describe the separation process in elution-extrusion counter-current chromatography (EECCC). As is known, EECCC consists of three steps: classical elution, sweeping elution, and extrusion. The perfect replacement approach means that during sweeping elution step, the mobile phase contained in the column moves and interacts with the "old" stationary phase in the same mode as during the classical elution step; the "new" and "old" stationary phases do not mix; and after the contacting with the mobile phase the concentration of solutes in the "old" stationary phase remains constant and this stationary phase volume is pushed ahead to the exit of the column. Equations are presented allowing the simulation of the chromatogram of solutes eluted from the column with the mobile phase during the elution period and the chromatogram of solutes pushed out of the column with the stationary phase during the extrusion period of EECCC. These equations can help to choose the optimal conditions for conducting elution-extrusion counter-current chromatography.     

Preparation and characterization of an immunoaffinity chromatography column for the selective extraction of trace contraceptive drug levonorgestrel from water samples

The preparation and characterization of an immunoaffinity chromatography (IAC) column for the specific extraction and enrichment of trace contraceptive drug levonorgestrel (LNG) from water samples were described. The IAC column was constructed by covalently coupling specific polyclonal antibody against LNG to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions, as well as the effect of flow rate on the extraction were carefully optimized. Pure water, 5% of methanol and 50% of methanol were respectively selected as loading, washing and eluting solutions, while the flow rates in the loading, washing and eluting steps were selected to be 1.0, 2.0 and 0.5 mL min⁻¹, respectively. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, extraction recovery and stability. It was found that, for IAC column packed with 0.2 g of solid support immobilized with antibody, the maximum capacity for LNG was about 260 ng. The extraction recoveries of the column for LNG at three different spiked concentrations were within 95.3-106.9%. After more than 35 times repeated usage, there was not significant loss of specific recognition. Using high performance liquid chromatography (HPLC) as an analytical tool, trace amount of LNG in the range of ng L⁻¹ was found in river water and wastewater samples after 600-fold enrichment, demonstrated the feasibility of the prepared IAC column for LNG extraction.     

Preparative isolation of bufalin and cinobufagin from Chinese traditional medicine ChanSu

In this investigation, a method is developed for the isolation and purification of bufadienolides (resibufogenin, cinobufagin, and bufalin) from the Chinese traditional medicine ChanSu. The crude ChanSu extract is prepared with solvent refluxing under optimal conditions. A new chromatographic approach for separating bufadienolides utilizes silica gel column chromatography with isocratic elution using cyclohexane-acetone (5:1) as the mobile phase to separate resibufogenin, followed by reversed-phase C18 preparative HPLC column using isocratic elution of methanol-water (72:28) to separate bufalin and cinobufagin. Combining these two methods, bufalin and cinobufagin could be completely separated in high purity and recovery, and the amounts obtained were 1.9 and 3.1 g, respectively, from 500 g of ChanSu. These two compounds have been identified by mass spectrometry and 1H NMR, and their purities were quantitated by HPLC at 99% and 98%, respectively.     

Synthesis of non-porous poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application in separation of biopolymers

The monodisperse, 5.0 μm non-porous poly(glycidylmethacrylate-co-ethylenedimethacrylate) (P_GMA/EDMA) beads were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. Based on this media, a weak cation exchange (WCX) stationary phase for high performance liquid chromatography (HPLC) was synthesized by a new chemical modification method. The prepared resin has advantages of biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The measured bioactivity recovery for lysozyme was 97 ± 5%. The dynamic protein loading capacity of the synthesized WCX packings was 20.5 mg/g. Four proteins were completely separated in 3.0 min using the synthesized WCX stationary phase. The experimental results show that the obtained WCX resin has very weak hydrophobicity. The WCX resin was also used for the rapid separation and purification of lysozyme from egg white in 3.0 min with only one step. The purity and specific bioactivity of the purified lysozyme was found more than 95% and 70.264 IU/mg, respectively.     

Monolith affinity chromatography for the rapid quantification of a single‐chain variable fragment immunotoxin

We developed a novel analytical method for concentration determination of tandem single‐chain antibody diphtheria toxin (immunotoxin). The method is based on polymethacrylate monoliths with Protein L ligands as the binding moiety. Different buffers were tested for elution of the Protein L‐bound immunotoxin and 4.5 M guanidinium hydrochloride performed best. We optimized the elution conditions and the method sequence resulting in a fast and robust method with a runtime <10 min. Fast determination of immunotoxin is critical if any process decisions rely on this data. We determined method performance and a lower limit of detection of 27 μg/mL and a lower limit of quantification of 90 μg/mL was achieved. The validity of the method in terms of residual analysis, precision, and repeatability was proven in a range from 100 to 375 μg/mL. The short runtime and ease of use of a high‐performance liquid chromatography method is especially useful for a process analytical tool approach. Bioprocesses related to immunotoxin where fermentation or other process parameters can be adjusted in accordance to the immunotoxin levels will be benefited from this method to achieve the highest possible purity and productivity.     

Chromatographic elution process design space development for the purification of saponins in Panax notoginseng extract using a probability‐based approach

A Monte Carlo method was used to develop the design space of a chromatographic elution process for the purification of saponins in Panax notoginseng extract. During this process, saponin recovery ratios, saponin purity, and elution productivity are determined as process critical quality attributes, and ethanol concentration, elution rate, and elution volume are identified as critical process parameters. Quadratic equations between process critical quality attributes and critical process parameters were established using response surface methodology. Then probability‐based design space was computed by calculating the prediction errors using Monte Carlo simulations. The influences of calculation parameters on computation results were investigated. The optimized calculation condition was as follows: calculation step length of 0.02, simulation times of 10 000, and a significance level value of 0.15 for adding or removing terms in a stepwise regression. Recommended normal operation region is located in ethanol concentration of 65.0–70.0%, elution rate of 1.7–2.0 bed volumes (BV)/h and elution volume of 3.0–3.6 BV. Verification experiments were carried out and the experimental values were in a good agreement with the predicted values. The application of present method is promising to develop a probability‐based design space for other botanical drug manufacturing process.     

开管毛细管亲和液相色谱初探

 提出了一种新的亲和色谱模式开管毛细管亲和液相色谱。在内径为 5 0 μm的毛细管内表面键合一层三嗪染料配体 ,利用毛细管电泳仪的压力系统进行亲和色谱实验。采用流动相切换洗脱技术 ,牛血清白蛋白和溶菌酶获得了有效的分离。连续运行 10次 ,溶菌酶的出峰时间、峰面积和峰高的相对标准偏差分别为0 1% ,4 3% ,3 7%。在 8 6ng～ 2 8 7ng范围内 ,溶菌酶的进样量与峰面积和峰高都呈线性关系 ,其相关系数分别为 0 9946和 0 9988。     

A Rapid Sample Preparation Method of Adriamycin from Plasma for HPLC Analysis

A rapid and comprehensive sample preparation method used to extract adriamycin from plasma has been investigated. The samples were passed through an Adsorbex extraction column with an elution solvent. The eluate was directly analyzed by reversed-phase high performance liguid chromatography (HPLC) with fluorescence detector. This solid/liquid extraction method is simpler than the conventional liquid/liquid extraction method. Different elution solvents were used to extract the adriamycin from samples with and without serum. The mobile phase was found to be the optimal elution solvent: 5 mM orthophosphoric acid 62.5% in methanol; acetonitrile; isopropanol = 15:15:7.5, pH 3.2. This method has the merits of better recovery (98%), higher reproducibility, and reguires shorter time and consumes less solvent.     

Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC)

This work aims at understanding the attachment mechanisms and stability of proteins on a chromatography medium to develop more efficient functionalization methodologies, which can be exploited in affinity chromatography. In particular, the study was focused on the understanding of the attachment mechanisms of bovine serum albumin (BSA), used as a ligand model, and protein G on novel amine-modified alumina monoliths as a stationary phase. Protein G was used to develop a column for antibody purification. The results showed that, at lower protein concentrations (i.e., 0.5 to 1.0 mg·mL⁻¹), protein attachment follows a 1st-order kinetics compatible with the presence of covalent binding between the monolith and the protein. At higher protein concentrations (i.e., up to 10 mg·mL⁻¹), the data preferably fit a 2nd-order kinetics. Such a change reflects a different mechanism in the protein attachment which, at higher concentrations, seems to be governed by physical adsorption resulting in a multilayered protein formation, due to the presence of ligand aggregates. The threshold condition for the prevalence of physical adsorption of BSA was found at a concentration higher than 1.0 mg·mL⁻¹. Based on this result, protein concentrations of 0.7 and 1.0 mg·mL⁻¹ were used for the functionalization of monoliths with protein G, allowing a maximum attachment of 1.43 mg of protein G/g of monolith. This column was then used for IgG binding–elution experiments, which resulted in an antibody attachment of 73.5% and, subsequently, elution of 86%, in acidic conditions. This proved the potential of the amine-functionalized monoliths for application in affinity chromatography.