非盐依赖层析的研究与应用

在非盐依赖层析操作中,吸附介质的成分、结构、密度等性质得到改进,所以料液离子强度的变化不会明显影响吸附 . 与常用的离子交换层析、疏水层析、亲硫层析等方法相比,该类技术能够降低对料液预处理的要求,提高蛋白质的稳定性,同时简化层析操作、降低纯化成本,具有大规模分离纯化蛋白质的潜力 . 近年来开发的多种非盐依赖层析介质与方法,都已在蛋白质纯化中得到应用 .     

Studies of lysozyme binding to histamine as a ligand for hydrophobic charge induction chromatography

Histamine was immobilized on Sepharose CL‐6B (Sepharose) for use as a ligand of hydrophobic charge induction chromatography (HCIC) of proteins. Lysozyme adsorption onto Histamine‐Sepharose (HA‐S) was studied by adsorption equilibrium and calorimetry to uncover the thermodynamic mechanism of the protein binding. In both the experiments, the influence of salt (ammonium sulfate and sodium sulfate) was examined. Adsorption isotherms showed that HA‐S exhibited a high salt tolerance in lysozyme adsorption. This property was well explained by the combined contributions of hydrophobic interaction and aromatic stacking. The isotherms were well fitted to the Langmuir equation, and the equilibrium parameters for lysozyme adsorption were obtained. In addition, thermodynamic parameters (ΔH_ads, ΔS_ads, and ΔG_ads) for the adsorption were obtained by isothermal titration calorimetry by titrating lysozyme solutions into the adsorbent suspension. Furthermore, free histamine was titrated into lysozyme solution in the same salt‐buffers. Compared with the binding of lysozyme to free histamine, lysozyme adsorption onto HA‐S was characterized by a less favorable ΔG_ads and an unfavorable ΔS_ads because histamine was covalently attached to Sepharose via a three‐carbon‐chain spacer. Consequently, the immobilized histamine could only associate with the residues on the protein surface rather than those in the hydrophobic pocket, causing a less favorable orientation between histamine and lysozyme. Further comparison of thermodynamic parameters indicated that the unfavorable ΔS_ads was offset by a favorable ΔH_ads, thus exhibiting typical enthalpy‐entropy compensation. Moreover, thermodynamic analyses indicated the importance of the dehydration of lysozyme molecule and HA‐S during the adsorption and a substantial conformational change of the protein during adsorption. The results have provided clear insights into the adsorption mechanisms of lysozyme onto the new HCIC material. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Molecular dynamics simulation and experimental validation of the effect of pH on protein desorption in hydrophobic charge induction chromatography

The ligands in hydrophobic charge induction chromatography (HCIC) are hydrophobic and ionisable. Thus, the pH is crucial for the separation performance in HCIC, especially for elution. However, it is difficult to obtain the microscopic information in HCIC through experimental means. In this work, molecular dynamics simulations are performed to examine the effect of pH on elution and protein conformational transition in HCIC, using a 46-bead β-barrel coarse-grained model protein and an HCIC adsorbent pore model constructed in an earlier work. Corresponding experiments are carried out for the validation of simulation results, using lysozyme and MEP Hypercel. Both the activities and fluorescence of lysozyme are examined to evaluate the conformational transition. The simulations indicate that the elution efficiency of protein increases with decreasing pH value in a non-linear manner. This is qualitatively consistent with the experimental results. MD simulations indicate that protein unfolding occurs in elution at all pH values. However, the experimental data show that the activity and conformation of lysozyme is independent of pH of the elution buffer. The microscopic information from simulation shows that protein unfolding is mainly observed on the adsorbent surface, but it cannot be detected in the experiments that only probe the proteins in the bulk liquid.     

Membrane chromatographic immunoassay method for rapid quantitative analysis of specific serum antibodies

This paper discusses a membrane chromatographic immunoassay method for rapid detection and quantitative analysis of specific serum antibodies. A type of polyvinylidine fluoride (PVDF) microfiltration membrane was used in the method for its ability to reversibly and specifically bind IgG antibodies from antiserum samples by hydrophobic interaction. Using this form of selective antibody binding and enrichment an affinity membrane with antigen binding ability was obtained in-situ. This was done by passing a pulse of diluted antiserum sample through a stack of microporous PVDF membranes. The affinity membrane thus formed was challenged with a pulse of antigen solution and the amount of antigen bound was accurately determined using chromatographic methods. The antigen binding correlated well with the antibody loading on the membrane. This method is direct, rapid and accurate, does not involve any chemical reaction, and uses very few reagents. Moreover, the same membrane could be repeatedly used for sequential immunoassays on account of the reversible nature of the antibody binding. Proof of concept of this method is provided using human hemoglobin as model antigen and rabbit antiserum against human hemoglobin as the antibody source.     

Purification of monoclonal antibodies by hydrophobic interaction chromatography under no-salt conditions

Hydrophobic interaction chromatography (HIC) is commonly used as a polishing step in monoclonal antibody purification processes. HIC offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. HIC, however, suffers from the limitation of use of high concentrations of kosmotropic salts to achieve the desired separation. These salts often pose a disposal concern in manufacturing facilities and at times can cause precipitation of the product. Here, we report an unconventional way of operating HIC in the flowthrough (FT) mode with no kosmotropic salt in the mobile phase. A very hydrophobic resin is selected as the stationary phase and the pH of the mobile phase is modulated to achieve the required selectivity. Under the pH conditions tested (pH 6.0 and below), antibodies typically become positively charged, which has an effect on its polarity and overall surface hydrophobicity. Optimum pH conditions were chosen under which the antibody product of interest flowed through while impurities such as aggregates and host cell proteins bound to the column. This strategy was tested with a panel of antibodies with varying pI and surface hydrophobicity. Performance was comparable to that observed using conventional HIC conditions with high salt.     

A modified IMAC method for the capture of target protein from mammalian cell culture harvest containing metal chelating species

Although immobilized metal affinity chromatography (IMAC) offers high capacity and protein selectivity it is not typically used commercially for the capture of native proteins from mammalian cell culture harvest. This is due mainly to the potential for low target recovery due to the presence of strong metal ion chelating species in the harvest that compete for the metal immobilized on the resin. To address this issue a buffer exchange step, such as tangential flow filtration (TFF), is added after harvest clarification and prior to IMAC to remove the interfering harvest components. The addition of a TFF step adds process time and cost and reduces target protein recovery. The elimination of the TFF might make IMAC competitive with other orthogonal methods of protein capture. In this study, we developed a modified IMAC method to allow the direct loading of clarified mammalian harvest without prior buffer exchange (direct IMAC). Although the target enzyme recovery was lower than that from standard IMAC the elimination of the buffer exchange step resulted in a 19% increase in overall enzyme recovery. The target enzyme capacity in direct IMAC was higher, in our experience, than the capacity of hydrophobic interaction (HIC) and ion-exchange (IEX) for protein capture. An economic evaluation of using direct IMAC as a capture step in manufacturing is also discussed.     

Computational tool for the early screening of monoclonal antibodies for their viscosities

Highly concentrated antibody solutions often exhibit high viscosities, which present a number of challenges for antibody-drug development, manufacturing and administration. The antibody sequence is a key determinant for high viscosity of highly concentrated solutions; therefore, a sequence- or structure-based tool that can identify highly viscous antibodies from their sequence would be effective in ensuring that only antibodies with low viscosity progress to the development phase. Here, we present a spatial charge map (SCM) tool that can accurately identify highly viscous antibodies from their sequence alone (using homology modeling to determine the 3-dimensional structures). The SCM tool has been extensively validated at 3 different organizations, and has proved successful in correctly identifying highly viscous antibodies. As a quantitative tool, SCM is amenable to high-throughput automated analysis, and can be effectively implemented during the antibody screening or engineering phase for the selection of low-viscosity antibodies.     

Generation of monospecific antibodies based on affinity capture of polyclonal antibodies

A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.     

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved.     

Characterisation of STREAMLINETM Phenyl

STREAMLINE Phenyl is a new hydrophobic interaction chromatography support designed for use in expanded bed adsorption. The phenyl groups are linked to STREAMLINE matrix via highly stable ether linkages. Within this development project the chemical and chromatographic stability as well as the breakthrough capacity for human IgG has been studied. The chemical stability was monitored as the carbon leakage from the matrix to the storage solution, pH 1–14 at 20 and 40 °C. The carbon content in the supernatant was determined with Total Organic Carbon (TOC) technique. In the chromatographic stability study STREAMLINE Phenyl was stored in eight different storage solutions under ambient conditions for 12 weeks and then tested in a chromatographic function test. The results show that the adsorbent is chemically stable and that the chromatographic properties are retained under the tested conditions. The breakthrough capacity study demonstrates the importance of the bed height for obtaining maximal dynamic capacity. Further, there is a good correlation between breakthrough data generated from packed bed and expanded bed runs.     

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

ABSTRACT

Immunoglobulin G–like bispecific antibodies with asymmetric architecture are among the most widely used bispecific antibody formats for diagnostic and therapeutic applications. The primary technical challenge for this format is how to achieve correctly paired assembly of four unique polypeptide chains. Advances in protein engineering and process development are being used to overcome these challenges and are driving a corresponding demand for sensitive analytical tools to monitor and control mispaired species. Here, we report a systematic approach for analysis and characterization of mispairing in asymmetric bispecific antibodies. This approach consists of three orthogonal components, the first of which is a liquid chromatography (LC)-mass spectrometry (MS)–based method to measure the mass of intact antibodies. This method is used for fast analysis of mispairing and requires minimal method development, which makes it an ideal choice for early-stage development. The second component is a hydrophobic interaction chromatography (HIC)–based mispairing method that is suitable for lot release testing. The HIC method is robust and quality control friendly, and offers great linearity, precision, and accuracy. The third component is a two-dimensional LC-MS method for on-line chromatographic peak identification, which not only expedites this task but also reduces the risk of undesirable modifications during conventional fraction collection. These three methods dovetail to form the foundation of a complementary toolbox for analysis and characterization of mispairing in asymmetric bispecific antibodies and provide guidance and support for process development throughout the drug development life cycle.     

A novel method for continuous chromatographic separation of monoclonal antibody charge variants by combining displacement mode chromatography and step elution

Charge heterogeneity of monoclonal antibodies is considered a critical quality attribute and hence needs to be monitored and controlled by the manufacturer. Typically, this is accomplished via separation of charge variants on cation exchange chromatography (CEX) using a pH or conductivity based linear gradient elution. Although an effective approach, this is challenging particularly during continuous processing as creation of linear gradient during continuous processing adds to process complexity and can lead to deviations in product quality upon slightest changes in gradient formation. Moreover, the long length of elution gradient along with the required peak fractionation makes process integration difficult. In this study, we propose a novel approach for separation of charge variants during continuous CEX chromatography by utilizing a combination of displacement mode chromatography and salt-based step elution. It has been demonstrated that while the displacement mode of chromatography enables control of acidic variants ≤26% in the CEX eluate, salt-based step gradient elution manages basic charge variant ≤25% in the CEX eluate. The proposed approach has been successfully demonstrated using feed materials with varying compositions. On comparing the designed strategy with 2-column concurrent (CC) chromatography, the resin specific productivity increased by 95% and resin utilization increased by 183% with recovery of main species >99%. Further, in order to showcase the amenability of the designed CEX method in continuous operation, the method was examined in our in-house continuous mAb platform.     

Establishment of a national network of validated and qualified laboratories for neutralizing anti-vaccinia antibodies titration

A Proficiency Testing Study (PTS) was organized in France by the French Health Products Safety Agency (Afssaps) aiming at assessing the performance of laboratories in performing a neutralizing anti-vaccinia antibodies titration method by plaque reduction neutralization test (PRNT). The ultimate goal was to establish a national network of qualified and validated laboratories. Five laboratories were included in the PTS and four submitted their data. Three samples of human sera containing various immunoglobulin concentrations (a "high" serum: s-576, a "medium" serum: Ref-19584 and a "low" serum: s-483) were tested by PRNT as described in a procedure supplied by Afssaps and developed in each laboratory during preliminary assays. Data were sent to Afssaps which performed the statistical analysis. The overall performance of the four participating laboratories was satisfactory. This allowed the four participating laboratories to be validated and then to be qualified by the Ministry of Health. Finally a national network for anti-vaccinia immunoglobulins titration was established.     

Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography

Therapeutic non-hinge-modified IgG4 molecules form bispecific hybrid antibodies with endogenous human IgG4 molecules via a process known as Fab-arm exchange (or called half molecule exchange). Analysis of the bispecific hybrids is critical for studies of half molecule exchange. A number of analytical methods are available to detect IgG4 hybrids. These methods mostly necessitate labeling or alteration of the model IgG4 molecules, or rely on time-consuming immunoassays and mass spectrometry. In addition, these methods do not allow isolation of hybrid antibodies. We report here the only analytical method to date that relies on chromatographic separation for detection of hybrids formed from intact antibodies in their native forms using pembrolizumab as an example. This method employs a mixed-mode chromatography using a Sepax Zenix SEC-300 column to separate a bispecific hybrid from the parental antibodies. The simultaneous quantitative monitoring of the newly formed hybrid and parental antibodies was achieved by UV absorption and/or protein fluorescence. The bispecific hybrid antibodies were purified with the same method for further biochemical characterization. The method has allowed monitoring of half molecule exchange between a human serum IgG4 and a tested IgG4 molecule, and has been implemented for the analysis of in vitro as well as in vivo samples.     

Synthesis and performance of 3D-megaporous structures for enzyme immobilization and protein capture

The preparation of megaporous bodies, with potential applications in biotechnology, was attempted by following several strategies. As a first step, naive and robust scaffolds were produced by polymerization of selected monomers in the presence of a highly soluble cross‐linker agent. Ion‐exchange function was incorporated by particle embedding, direct chemical synthesis, or radiation‐induced grafting. The total ionic capacity of such systems was 1.5 mmol H⁺/g, 1.4 mmol H⁺/g, and 17 mmol H⁺/g, respectively. These values were in agreement with the ability to bind model proteins: observed dynamic binding capacity at 50% breakthrough was ?7.2 mg bovine serum albumin/g, ?7.4 hen egg‐white lysozyme (HEWL) mg/g, and ?108 HEWL mg/g. In the later case, total (static) binding capacity reached 220 mg/g. It was observed that the structure and size of the megapores remained unaffected by the grafting procedure which, however, allowed for the highest protein binding capacity. Lysozyme supported on grafted body showed extensive clarification activity against a Micrococcus lysodekticus suspension in the flow‐through mode, i.e., 90% destruction of suspended microbial cells was obtained with a residence time ≈ 18 min. Both protein capture and biocatalysis applications are conceivable with the 3D‐megaporous materials described in this work. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Increase in synthesis of human monoclonal antibodies by transfected Sp2/0 myeloma mouse cell line under conditions of microgravity

Microgravity can influence cell growth and function. A transfected Sp2/0 myeloma cell line P3A2 producing a human IgG1 anti-TNF monoclonal antibody was cultivated in static culture, spinner flasks and simulated microgravity using a rotating wall vessel bioreactor. Microgravity significantly decreased cell growth (from 1.7×10⁶ to 7.9×10⁵ cells/ml), but facilitated the synthesis of antibodies, (1.8, 1.3 and 0.5 g of anti-TNF hmAb per 10⁶ viable cells for cells cultivated under microgravity, in spinner flasks and static cultures, respectively). The results suggest that microgravity could be applied to improve the specific productivity of cell lines producing potentially important therapeutic proteins.     

RNA oligoprobe capture RT-PCR, a sensitive method for the detection ofGrapevine fanleaf virus in Tunisian grapevines

A simple, sensitive and specific RNA capture method is described for the detection of Grapevine fanleaf virus (GFLV) in infected grapevines. This method consists of hybridizing GFLV-RNAs to oligoprobes immobilized on nylon membranes, followed by RT-PCR amplification of targeted viral sequences. The RNA oligoprobe capture RT-PCR method is 10-fold more sensitive than IC-RT-PCR. The efficiency of the RNA oligoprobe capture RT-PCR and the reuse of immobilized oligoprobe membranes without loss of efficiency could make this procedure suitable for the routine diagnosis of GFLV in grapevines.     

Purification of monomeric mAb from associated aggregates using selective desorption chromatography in hydroxyapatite systems

Selective desorption on ceramic hydroxyapatite (CHT) was implemented for the purification of monomeric monoclonal antibody (mAb) from associated aggregates and other post-protein A step impurities. A robotic liquid handling system was employed to carry out a parallel batch screen of selective desorbents on a post-protein A step mAb mixture. The effect of different phosphate concentrations was also investigated. Selective batch separations were achieved between monomeric mAb and associated aggregates/impurities. The batch screen results also established optimal mobile phase conditions for each selective desorbent. These initial batch results were then used to guide column separations, and baseline separation of monomeric mAb from associated aggregates and impurities was achieved, validating the screening results. Selective desorption also resulted in improved separations on CHT, with 100% yield of pure monomeric mAb as compared to 61% and 79%, respectively, for conventional linear and step gradient operations. This proof of concept study demonstrates selective desorption on CHT as an effective separation technique for the purification of monomeric mAb from associated aggregates and other post-protein A step impurities in a single process step.