Fractionation of the human plasma proteome for monoclonal antibody proteomics-based biomarker discovery

mAb proteomics, a reversed biomarker discovery approach, is a novel methodology to recognize the proteins of biomarker potential, but requires subsequent antigen identification steps. While in case of high-abundant proteins, it generally does not represent a problem, for medium or lower abundant proteins, the identification step requires a large amount of sample to assure the proper amount of antigen for the ID process. In this article, we report on the use of combined chromatographic and precipitation techniques to generate a large set of fractions representing the human plasma proteome, referred to as the Analyte Library, with the goal to use the relevant library fractions for antigen identification in conjunction with mAb proteomics. Starting from 500?mL normal pooled human plasma, this process resulted in 783 fractions with the average protein concentration of 1?mg/mL. First, the serum albumin and immunoglobulins were depleted followed by prefractionation by ammonium sulfate precipitation steps. Each precipitate was then separated by size exclusion chromatography, followed by cation and anion exchange chromatography. The 20 most concentrated ion exchange chromatography fractions were further separated by hydrophobic interaction chromatography. All chromatography and precipitation steps were carefully designed aiming to maintain the native forms of the intact proteins throughout the fractionation process. The separation route of vitamin D-binding protein (an antibody proteomics lead) was followed in all major fractionation levels by dot blot assay in order to identify the library fraction it accumulated in and the identity of the antigen was verified by Western blot.     

Effect of α-Ketoglutarate on Monoclonal Antibody Production of Hybridoma Cell Lines in Serum-Free and Serum-Containing Medium

Process development and optimization for increase population growth and protein productivity in mammalian cell culture have been studied for many years. In this study, the behavior of hybridoma cells was investigated using six-well micro-titer plate systems with a working volume of 4 ml. Mouse hybridoma cell lines D2 and 2C83G2 were seeded in serum-free and serum-containing media and cultured for 8 days. alpha-Ketoglutarate is an integral component of the tricarboxylic acid (TCA) cycle and is produced from glutamine via glutamate. To study its effect on cell growth, metabolism, and monoclonal antibody (mAb) production, 2 mM alpha-ketoglutarate (pH 7.2) was added in both media at the beginning of the cultivation and in another set after 72 h. High cell density was observed in D2 cell culturing in serum-free medium, while 2C83G2 cell line showed high cell density in serum-containing medium. However, both cell lines cultured in serum-free medium gave viability above 70% when grown for 8 days. The supplement of 2 mM alpha-ketoglutarate supported cell growth and mAb production of both hybridoma cell lines in serum-free and serum-containing medium. The addition of alpha-ketoglutarate at the beginning of the batch cultivation gave better result in cell growth and mAb production as compared to alpha-ketoglutarate supplementation after 72 h. However, addition after 72 h was better than no addition at all. This indicates that alpha-ketoglutarate have a positive effect on production and release of antibody.     

Design of experiments for micellar electrokinetic chromatography method development for the monitoring of water-soluble vitamins in cell culture medium

Biopharmaceutical production takes place in complex processes which should be thoroughly understood. Therefore, the iConsensus project focuses on developing a monitoring platform integrating several process analytical technology tools for integrated, automated monitoring of the biopharmaceutical process. Water-soluble vitamin monitoring using (microchip) capillary electrophoresis (CE) is part of this platform. This work comprises the development of conventional CE methods as the first part towards integrated vitamin monitoring. The vitamins were divided based on their physical–chemical properties to develop two robust methods. Previously, a method for the analysis of cationic vitamins (pyridoxine, pyridoxal, pyridoxamine, thiamine and nicotinamide) in cell culture medium was developed. This work focused on the development of a micellar electrokinetic chromatography method for anionic and neutral vitamins (riboflavin, d -calcium pantothenate, biotin, folic acid, cyanocobalamin and ascorbic acid). By employing multivariate design of experiments, the background electrolyte (BGE) could be optimised within one experiment testing only 11 BGEs. The optimised BGE conditions were 200 mM borate with 77 mM sodium dodecyl sulphate at a pH of 8.6. Using this BGE, all above-mentioned cationic, anionic and neutral vitamins could be separated in clean samples. In cell culture medium, most anionic and neutral vitamins could be separated. Combining the two methods allows for analysis of cationic, anionic and neutral vitamins in cell culture medium samples. The next step towards integrated vitamin monitoring includes transfer to microchip CE. Due to the lack of fast and reliable methods for vitamin monitoring, the developed capillary methods could be valuable as stand-alone at-line process analytical technology solutions as well.     

Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography

Humanized monoclonal antibodies (mAbs) hold significant promise as biopharmaceuticals. One of the main challenges faced in the purification of mAbs is their separation from bovine serum albumin, which is the main protein present in most mammalian cell culture media. This paper discusses the purification of humanized mAb hIgG1-CD4 from CHO cell culture media by hydrophobic interaction membrane chromatography using a stack of microporous synthetic membranes. The effects of solution conditions on mAb solubility and binding on the membrane were first studied. The separation of a simulated mixture of bovine albumin and the mAb was then carried out to examine the feasibility of mAb purification. Separation experiments carried out under optimized conditions demonstrated that this membrane-based technique could be used for mAb purification from cell culture media. High purity (97%) and recovery (in excess of 97%) were obtained.     

Model simulation and experimental verification of a cation-exchange IgG capture step in batch and continuous chromatography

The cation-exchange capture step of a monoclonal antibody (mAb) purification process using single column batch and multicolumn continuous chromatography (MCSGP) was modeled with a lumped kinetic model. Model parameters were experimentally determined under analytical and preparative conditions: porosities, retention factors and mass transfer parameters of purified mAb were obtained through a systematic procedure based on retention time measurements. The saturation capacity was determined through peak fitting assuming a Langmuir-type adsorption isotherm. The model was validated using linear batch gradient elutions. In addition, the model was used to simulate the start-up, cyclic steady state and shut down behavior of the continuous capture process (MCSGP) and to predict performance parameters. The obtained results were validated by comparison with suitable experiments using an industrial cell culture supernatant. Although the model was not capable of delivering quantitative information of the product purity, it proved high accuracy in the prediction of product concentrations and yield with an error of less than 6%, making it a very useful tool in process development.     

Evaluation of a PEG/hydroxypropyl starch aqueous two‐phase system for the separation of monoclonal antibodies from cell culture supernatant

In this study, an aqueous two‐phase system (ATPS) with PEG and hydroxypropyl starch (HPS) was used to separate monoclonal antibody (mAb) from Chinese hamster ovary cell culture supernatant. The phase diagram of the PEG/HPS ATPS was determined, and the effects of NaCl addition were investigated. The results showed that NaCl addition could lead to a shift of the binodal curve and that phase separation would occur at higher PEG and HPS concentrations. The effects of NaCl addition, pH, and the load of cell supernatant on the partitioning of mAb in a PEG/HPS ATPS were investigated. It was found that with 6% cell supernatant and 15% NaCl addition at pH 6.0, the yield of mAb in the upper phase was 96.7% with a purity of 96.0%. The back‐extraction of mAb with a PEG/phosphate ATPS were also studied, and the results showed that after the two‐step extraction with ATPSs the purity of mAb could reach 97.6 ± 0.5% with a yield of 86.8 ± 1.0%, which was comparable to the purification with Protein A chromatography. These results indicate that the two‐step extraction with PEG/HPS and PEG/phosphate ATPSs might be a promising alternative for the separation of mAb from cell culture supernatant.     

Porous composite scaffolds of hydroxyapatite/silk fibroin via two‐step method

A two‐step method was used to fabricate the hydroxyapatite (HAP)/silk fibroin (SF) scaffolds, i.e. the nano‐sized HAP/SF composite powders were prepared by co‐precipitation, which were then blended with SF solution to fabricate the HAP/SF composite scaffolds. The obtained scaffolds showed a 3D porous structure. The porosity was higher than 90% with the average macropore size of 214.2 µm. Moreover, the nano‐sized HAP/SF composite powders were uniformly dispersed in the silk fibroin matrix, which provided the scaffolds enhanced compressive properties. The cell culture assay showed that the scaffolds fabricated by the two‐step method could improve the cell proliferation and osteogenic differentiation when compared with those prepared by the conventional one‐step blending method. The results suggested that the two‐step method could promote the uniform dispersion of HAP in the SF matrix and efficient combination between the HAP and the matrix, which may provide a potential application in the composite scaffold preparation for tissue engineering. Copyright © 2009 John Wiley & Sons, Ltd.     

LC-HRMS-based targeted metabolomics for high-throughput and quantitative analysis of 21 growth inhibition-related metabolites in Chinese hamster ovary cell fed-batch cultures

Chinese hamster ovary (CHO) cells have been widely used in the biopharmaceutical industry for production of therapeutic proteins. CHO cells in fed-batch cultures produce various amino acid–derived intermediate metabolites. These small molecule metabolic byproducts have proven to be critical to cell growth, culture performance, and, more interestingly, antibody drug productivity. Herein, we developed an LC-HRMS-based targeted metabolomics approach for comprehensive quantification of total 21 growth inhibition-related metabolites generated from 14 different amino acids in CHO cell fed-batch cultures. High throughput derivatization procedures, matrix-matched calibration curves, stable isotope-labeled internal standards, and accurate mass full MS scan were utilized to achieve our goal for a wide range of metabolite screening as well as validity and reliability of metabolite quantification. We further present a novel analytical strategy for extending the assay's dynamic range by utilizing naturally occurring isotope M + 1 ion as a quantification analog in the circumstances where the principal M ion is beyond its calibration range. The integrated method was qualified for selectivity, sensitivity, linearity, accuracy, precision, isotope analysis, and other analytical aspects to demonstrate assay robustness. We then applied this metabolomics approach to characterize metabolites of interest in a CHO cell-based monoclonal antibody (mAb) production process with fed-batch bioreactor culture mode. Absolute quantification combined with multivariate statistical analysis illustrated that our target analytes derived from amino acids, especially from branched-chain amino acids, closely correlated with cell viability and significantly differentiated cellular stages in production process.     

Estimation of the optimal concentrations of residual sugar and cell growth rate for a fed-batch culture ofSaccharomyces cerevisiae

Estimation of the optimal concentrations of residual sugar in medium for a fed-batch culture of Baker’s yeast has been studied and practiced. The concentrations, however, depended on different species and targets of the biomass, which was expected to be made. Kinetic changes of the residual phosphate salt in the medium conformed to a logarithmic process until the fourth hour during an 11-h culture. The parabolic method (see ref. 9 later in article) might be qualified to maintain the concentrations of residual sugar around 0.15 g/L. It was demonstrated that cell growth followed a sigmoid process during a fed-batch culture, because the cells consumed the nutrient with two metabolic pathways, one was for cell conversion and another was for non-cell conversion. With the parabolic method, we can estimate kinetics of cell growth and cell growth rate during the culture.     

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.     

Determination of iodine in biological materials by neutron activation analysis

A method of determination of iodine (total and PBI) in serum, urine and other biological materials has been developed. The method consists in a gamma-spectrometric measurement of¹²⁸I activity after its radiochemical separation. The radiochemical separation procedure includes wet decomposition of the samples in a nitric acid medium followed by a few separation steps, the essential step being the substoichiometric extraction of iodide with a chloroform solution of tetraphenylarsonium chloride. Owing to the application of the substoichiometric separation, a high radiochemical purity of the separated iodine is achieved and the determination of the yield of radiochemical separation is not necessary.     

Laser flash photolysis study of the photocatalytic step of the photo-fenton reaction in saline solution

The photo-Fenton reaction (Fe2+/Fe3+, H2O2, UV light) is strongly inhibited by high concentrations of added chloride ion. In this work, the effect of added chloride ion on the photocatalytic step that converts Fe(III) back to Fe(II) is studied by nanosecond laser flash photolysis over a wide range of pH (1.0-3.3) and concentrations of Fe(III) (0.1-1.0 mM) and chloride ion (0.05-0.75 M). An explicit mechanistic model based on the preferential formation of the less-reactive Cl2*- radical anion via two routes (competitive photolysis of the iron(III)-chloride complex to chlorine atoms instead of the desired hydroxyl radical and pH-dependent scavenging of the hydroxyl radical by chloride ion) is proposed. This model, which fits the laser flash photolysis data for the production and decay of Cl2*- over the entire range of conditions investigated, suggests that inhibition of the photocatalytic step of the photo-Fenton process in the presence of chloride ion can be circumvented by maintaining the pH of the medium at or slightly above 3.0 throughout the reaction.     

Laboratory Production of Human Prolactin from CHO Cells Adapted to Serum-Free Suspension Culture

Human prolactin (hPRL) is a polypeptide with 199 amino acids and a molecular mass of 23?kDa. Previously, a eukaryotic hPRL expression vector was used to transfect Chinese hamster ovary (CHO) cells: this work describes a fast and practical laboratory adaptation of these transfected cells, in ~40?days, to grow in suspension in serum-free medium. High cell densities of up to 4.0?×?10⁶ cell/ml were obtained from spinner flask cultures and a stable and continuous production process was developed for at least 30?days. Two harvesting strategies were set up, 50 or 100?% of the total conditioned medium being collected daily and replaced by fresh culture medium. The volumetric productivity was 5?C7???g hPRL/ml, as determined directly in the collected medium via reversed-phase HPLC (RP-HPLC). A two-step process based on a cationic exchanger followed by size exclusion chromatography was applied to obtain purified hPRL from conditioned medium. Two hPRL isoforms, non-glycosylated and glycosylated, could also be separated by high-performance size-exclusion chromatography (HPSEC) and, when analyzed by RP-HPLC, HPSEC, Western blotting, and bioassay, were found to be comparable to the World Health Organization International Reference Reagent of hPRL. These results are useful for the practical scale-up to the pilot and industrial scale of a bioprocess based on CHO cell culture.     

Synthesis of quinoline thioethers as novel small molecule enhancers of monoclonal antibody production in mammalian cell culture

A small library of novel quinoline derivatives containing different thioether substituents at position 4 have been synthesized and screened in murine hybridoma cell culture for their ability to enhance monoclonal antibody (mAb) production. From this set of compounds, four compounds have been discovered that enhanced immunoglobulin G (IgG) titer over negative control cultures due to an increase in specific productivity. These results demonstrate the utility of using organic synthesis and parallel ligand screening methods to discover novel cell culture additives that may be useful for increasing mAb yield in industrial biomanufacturing processes.     

Electrochemical determination of femtomole amounts of free reduced and oxidized glutathione. Application to human hair follicles.

A cost-efficient process was specifically designed for the preparation of gram amounts of highly pure murine immunoglobulin (Ig) G₁ monoclonal antibodies (mAbs). This rapid, simple and scalable purification process employs a unique binding and elution protocol for IgG₁ mAbs on a silica-based, mixed-mode ion-exchange resin followed by conventional anion-exchange chromatography. mAbs are bound to BakerBond ABx medium at pH 5.6 directly from serum-supplemented hybridoma culture supernatants. Contamining proteins and nucleic acids are removed by an intermediate wash at pH 6.5, followed by the specific elution of IgG₁ mabs with 100 mM Tris-HCL (pH 8.5). The mAb eluate is then loaded directly on to QAE-Sepharose Fast Flow medium and eluted with 10 mM sodium phosphate buffer (pH 7.4), containing 150 mM sodium chloride. The resulting IgG₁ mAbs are greater than 98% pure, free from measurable endotoxin, formulated in a physiological buffer and suitable for in vivo applications.     

Clinical grade lentiviral vector purification and quality control requirements

Lentiviral vectors have been proven to be a powerful tool in gene therapies that includes the ability to perform long-term gene editing in both dividing and non-dividing cells. In order to meet the rising demand for clinical-grade lentiviral vectors for future clinical trials and requirements by regulatory agencies, new methods and technologies were developed, including the rapid optimization of production and purification processes. However, gaps still exist in achieving ideal yields and recovery rates in large-scale manufacturing process steps. The downstream purification process is a critical step required to obtain a sufficient quantity and high-quality lentiviral vectors products, which is challenged by the low stability of the lentiviral vector particles and large production volumes associated with the manufacturing process. This review summarizes the most recent and promising technologies and enhancements used in the large-scale purification process step of lentiviral vector manufacturing and aims to provide a significant contribution towards the achievement of providing sufficient quantity and quality of lentiviral vectors in scalable processes.     

Nickel determination in osteoblast-like cell culture medium by adsorptive cathodic stripping voltammetry with a mercury microelectrode

The purpose of this study is to investigate the influence of nickel, which is an alloying element in commonly used metallic biomaterials, on the biomaterials mineralization process. An electrochemical method was developed to quantify this metal ion in osteoblast-like cell culture medium (OST) by performing adsorptive cathodic stripping voltammetry (CSV) with dimethylglyoxime (DMG) at a mercury film microelectrode (MFM). The optimized analytical conditions and the square-wave CSV parameters for the analysis are: DMG concentration: 5.00 × 10⁻⁴ mol L⁻¹; ammonium chloride buffer: 0.10 mol L⁻¹ (pH 9.2); frequency: 50 Hz, amplitude 20 mV; step: 2 mV; adsorption time: 10 s, deposition potential: −0.70 V and reduction potential: −1.20 V. The limit of detection was 7.70 × 10⁻⁹ mol L⁻¹ for an adsorption time of 10 s. The results achieved by CSV using the MFM were compared to those obtained by atomic absorption spectrometry (AAS) to ensure the reliability of the electrochemical method. The mineralization process was evaluated by biochemical and histochemical assays.     

Contribution of cell culture additives to the two-dimensional protein patterns of mouse macrophages

Low levels of fetal calf serum (FCS), used as protein supplement in cell culture medium, were traced in preparations of primary murine macrophages (bone-marrow-derived macrophages (BMM) and peritoneal macrophages (PM)). Main components of this common additive were mapped in 2-DE by means of differential image gel electrophoresis and immunoblotting. Additional washing steps in cell preparation helped to decrease the levels of the four highest abundance foetal serum proteins (serum albumin (SA), alpha1-fetoprotein (AFP), alpha1-antitrypsin (alpha1AT) and transferrin (Tf)) to less than 1% of total protein. Macrophage spot pattern was recorded in parallel and showed little variation. Results presented are supposed to be of general interest for cell preparations with similar background.     

高效光解水光电极设计的研究进展（英文）

光电化学(PEC)分解水过程被认为是由太阳能制氢的一个有前景的路径,PEC的关键在于高效电极的设计.最近的十多年里有关材料设计、共催化剂研究和电极制造取得了重大进展,但仍存在一些关键挑战尚未解决,包括迫切所需的转化效率.作为PEC过程的三个关键步骤:光采集、电荷转移和表面反应,发生在很广的时间尺度(10~(–12)–10~0 s)内,如何组织好这一连串的步骤以促进各步骤间的无缝协作从而实现高效的PEC过程显得非常重要.基于高效稳定PEC光电极设计的研究进展,本文重点综述了整体考虑的三个主要标准,总结了一些基本原则和潜在的策略,尤其讨论了挑战与前景.     

Oxidation Mechanism of Fluorescein at Glassy Carbon Electrode

This study investigates redox properties of fluorescein (FLSC), a fluorescent tracer with many applications in several areas, markedly in biochemical research and health care diagnosis, on glassy carbon electrode (GCE) at a wide interval of pH by using voltammetric techniques. Three peaks were observed at different potentials. The investigation revealed that FLSC is irreversibly electroxidized under a diffusion‐controled and pH–dependent process. The oxidation process in acid and physiological media occurs in two consecutive steps with formation of a main electroactive oxidation product in acid medium. Both oxidation steps involve the transfer of one electron and one proton, corresponding to the oxidation of phenolic groups with formation of ortho‐quinone derivatives, which are reversibly reduced to form catechol derivatives, and/or polymeric products. One electron and one proton are removed from the phenolic group at the position C6’ at the first step and at position C3’ at the second step. The diffusion coefficient of FLSC was assessed in pH=7.0 phosphate buffer (9.77×10⁻⁵ cm² s⁻¹). A differential pulse voltammetric method for determination of FLSC in physiological medium was also proposed.