Synthesis, properties, and reactivity of cocaine benzoylthio ester possessing the cocaine absolute configuration

One aspect of immunopharmacotherapy for cocaine abuse involves the use of a catalytic monoclonal antibody (mAb) to degrade cocaine via hydrolysis of the benzoate ester. A cocaine benzoylthio ester analogue provides a means to implement high-throughput selection strategies to potentially isolate mAbs with high activity. The required analogue was synthesized starting from (-)-cocaine hydrochloride and possessed the cocaine absolute configuration. Key points in the preparation were the introduction of the sulfur atom at C-3 via a bromomagnesium thiolate addition to the exo face of anhydroecgonine, separation of C-2 diastereomers, recycling of a C-2 thio ester byproduct, and formation of the necessary C-2 methyl and C-3 benzoylthio esters. Effects resulting from the lower electronegativity and greater hydrophobicity of sulfur compared to oxygen were observed. These characteristics could result in interesting drug properties. Furthermore, the analogue was found to be a substrate for catalytic mAbs that hydrolyze cocaine as monitored by HPLC and also spectrophotometry by coupling cleavage of the benzoylthio ester to the disulfide exchange with Ellman's reagent. Screening antibody libraries with the new cocaine analogue using the spectroscopic assay provides an avenue for the high-throughput identification of catalysts that efficiently breakdown cocaine.     

Development of a two-step chromatography procedure that allows the purification of a high-purity anti-histone H1 monoclonal immunoglobulin M antibody with immunosuppressant activity

In organ transplantation, the development of a novel immunosuppressant free of the need for permanent administration and any serious side effects has eagerly been awaited. We have previously reported that an anti-histone H1 polyclonal antibody has immunosuppressant activity. Here we prepared an anti-histone H1 monoclonal antibody as an analytical tool to elucidate its mechanism of immunosuppression. The isotype of this monoclonal antibody was immunoglobulin M. A monoclonal antibody prepared for administration to organ transplantation model animals should not contain any allogenic proteins and should have high purity. Therefore, we conducted a two-step chromatography procedure, consisting of strong anion-exchange chromatography and gel filtration chromatography, to purify an anti-histone H1 monoclonal immunoglobulin M antibody from the serum-free culture supernatant of hybridomas. Consequently, we successfully purified the monoclonal antibody at 96%, a purification rate at which its administration to organ transplantation model animals is possible.     

A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens

Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 microg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.     

Fractionation of the human plasma proteome for monoclonal antibody proteomics‐based biomarker discovery 2: Antigen identification by dot‐blot array screening

Immunization with complex mixtures, like the human plasma resulted in the generation of cloned mAb libraries (PlasmaScan? and QuantiPlasma? libraries, with >1000 individual mAbs) reacting with a nonredundant set of antigenic epitopes. mAb proteomics refers to quasi‐hypothesis‐free profiling of plasma samples with nascent or cloned mAb libraries for the discovery of disease‐specific biomarkers. Once mAbs with biomarker potential have been identified, the next task is the determination of cognate antigens recognized by the respective mAbs. To determine the cognate protein antigen corresponding to each individual mAbs in the cloned mAb libraries, we have separated human plasma by consecutive steps of desalting and various chromatography procedures. The process resulted in 783 fractions, which we termed “Analyte Library” (AL). The AL represents the human plasma proteome in relatively low‐protein complexity fractions. Here, to determine the utility of the AL, we selected ten plasma proteins and checked for their presence in the fractions. Among the ten cases, the distribution of four selected plasma proteins matched expectations, as these proteins were present only in a few fractions corresponding to their physical, chemical, and biochemical properties. However, in six cases, we observed “smear” ‐like distribution or complete absence of the proteins, suggesting that protein–protein interactions or protein variants may alter the observed plasma distribution profiles. Nevertheless, we conclude that the AL is an efficient, high throughput tool to complement the mAb biomarker discovery process with cognate protein antigen identification for each mAbs.     

DNA spike studies for demonstrating improved clearance on chromatographic media

DNA spike clearance methods were used to demonstrate improved clearance factors on anion exchange and hydrophobic interaction columns used in the production of human therapeutic proteins. DNA clearance at large-scale was first measured for a monoclonal antibody expressed in Chinese Hamster Ovary (CHO) cells and an antibody fragment expressed in Escherichia coli. Small-scale spike experiments were then performed on individual chromatographic steps using host-specific DNA paired with TaqMan PCR assay methods. This approach has advantages of improved specificity, sensitivity, cost and throughput compared to other types of spike clearance methods. The anion exchange column used in the monoclonal antibody process was shown to have very high capacity for CHO DNA, resulting in greater than 7.1 log reduction. The anion exchange and hydrophobic interaction columns used in the antibody fragment process were shown to have high E. coli DNA clearance capability, with greater than 5.1 and 5.3 logs clearance, respectively. Compared to the large-scale process, higher log reduction values were achieved in small-scale spike clearance studies by challenging the chromatographic steps with load DNA levels 2–5 logs higher than the large-scale process levels. Using highly specific and sensitive spike clearance methods, we demonstrated consistently high DNA clearance factors for each of the production processes that meet industry and regulatory standards for human therapeutics. The method is applicable to a broad range of industrial scale processes where demonstration of the robustness of DNA clearance is necessary to support development or licensure of biopharmaceutical products.     

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.     

Purification of monoclonal antibody from tobacco extract using membrane-based bioseparation techniques

Transgenic plants offer a promising system for large-scale production of therapeutic proteins such as monoclonal antibodies (mAbs). This paper describes a membrane-based process suitable for purification of a humanized mAb expressed in tobacco. Most monoclonal antibody purification schemes rely on the use of Protein A as the affinity ligand for antibody capture. The main objective of our work was to develop non-Protein A-based purification methods to avoid some of the problems and limitations associated with this ligand, e.g. cost, immunotoxicity, and antibody aggregation during elution. Ion exchange membrane chromatography (IEMC) was used for primary capture and preliminary purification of the mAb from tobacco juice. Hydrophobic interaction membrane chromatography (HIMC) was then used for high-resolution purification, followed by ultrafiltration for polishing, desalting and buffer exchange. Using this scheme, both high mAb purity (single peak in size exclusion chromatogram, i.e., ca. 100% purity) and high recovery (77% of mAb spiked into the tobacco extract) were achieved. Membrane chromatography is generally considered unsuitable for resolving bound proteins by gradient elution and is therefore commonly used in the bind and elute mode with a single-step change of mobile phase. We show that the gradient elution process in the HIMC step can be optimized to increase the resolution and thereby obtain product of high purity.     

A universal surrogate peptide to enable LC-MS/MS bioanalysis of a diversity of human monoclonal antibody and human Fc-fusion protein drug candidates in pre-clinical animal studies

For the development of human antibody Fc (fraction crystallizable) region-containing therapeutic protein candidates, which can be either monoclonal antibodies (mAbs) or pharmacologically active proteins/peptides fused to the Fc region of human Immunoglobulin G (IgG), reliable quantification of these proteins in animal pharmacokinetic study plasma samples is critical. LC-MS/MS has emerged as a promising assay platform for this purpose. LC-MS/MS assays used for bioanalysis of human antibody Fc region-containing therapeutic protein candidates frequently rely upon quantification of a 'signature' surrogate peptide whose sequence is unique to the protein analyte of interest. One drawback of the signature peptide approach is that a new LC-MS/MS assay must be developed for each new human Fc region-containing therapeutic protein. To address this issue, we propose an alternative 'universal surrogate peptide' approach for the quantification of human antibody Fc region-containing therapeutic protein candidates in plasma samples from all nonclinical species. A single surrogate tryptic peptide was identified in the Fc region of most human antibody Fc-containing therapeutic protein candidates. An LC-MS-MS method based upon this peptide was shown to be capable of supporting bioanalysis of a diversity of human Fc region-containing therapeutic protein candidates in plasma samples of all commonly used animal species.     

A high-throughput microchip-based glycan screening assay for antibody cell culture samples

A high-throughput screening assay was developed to quantify major glycan species in the crude mammalian cell culture samples for monoclonal antibodies (mAbs). This method utilizes high-speed microchip electrophoresis separation following a fast sample preparation procedure. Using a 96-well ultra-filtration membrane, interfering species in the cell culture media were efficiently removed as the samples were concentrated. A commercial microchip electrophoresis instrument was used for high-speed separation, allowing each sample to be analyzed in less than 1 min. This method is well suited for the purpose of high-throughput antibody glycan profiling during cell culture expression, including clone selection and cell culture process optimization. The relative levels of high mannose (HM), fucosylated and galactosylated glycan species in the Fc domain can be determined for hundreds of crude cell culture samples in a few hours.     

Spatially addressable protein array: ssDNA-directed assembly for antibody microarray

Protein microarray offers a means for high-throughput profiling of cellular proteins to provide insights into the mechanisms of biological processes. This study describes the design and fabrication of a robust platform, spatially addressable protein array (SAPA), by exploring the specificity of ssDNA hybridization for self-assembly of semi-synthetic ssDNA-antibody conjugates which capture antigens from complex biological samples. This approach does not involve the direct immobilization of antibodies nor antigen, but instead captures the target antigens in the solution phase followed by self-directed assembly of the complex onto the surface. In an effort to optimize the platform, the effects of surface chemistry, nonspecific protein adsorption, facile preparation, and purification of ssDNA-conjugated antibody and capture of the antigen from a complex biological sample such as cell lysate were examined. This platform allowed antigen detection in cell lysate with high sensitivity (1 pM). The method described herein can be extended to the high-throughput detection of other interacting molecules in solution phase and their subsequent assembly onto any substrate.     

Hydrophobic charge‐induction resin with 5‐aminobenzimidazol as the functional ligand: preparation,protein adsorption and immunoglobulin G purification

A new hydrophobic charge‐induction chromatography resin was prepared with 5‐aminobenzimidazol as functional ligand and polyacrylic ester beads as matrix. Adsorption isotherms and adsorption in columns were investigated using human immunoglobulin G and bovine serum albumin as model proteins, and the influence of pH and NaCl concentration was discussed. Results showed that the ligand density was 195 μmol/mL gel, and protein selectivity can be improved by controlling pH and salt addition. An optimized purification process (sample loading at pH 8.0 with 0.2 M NaCl and elution at pH 5.0) was performed to purify human immunoglobulin G from bovine serum albumin containing feedstock, which resulted in human immunoglobulin G purity of 99.7% and recovery of 94.6%. A similar process was applied for the purification of monoclonal antibody from cell culture supernatant, which showed antibody purity of 94.9% and recovery of 92.5%. The results indicated that the new resin developed had comparable performance as Protein A chromatography and would be suitable for antibody purification from complex feedstock.     

Purifying the masses: integrating prepurification quality control, high-throughput LC/MS purification, and compound plating to feed high-throughput screening

In this paper we report using a parallel, four-channel HPLC/MUX/MS purification system, the Purification Factory, to purify thousands of compounds destined for high-throughput screening in a single month. The maximum sample throughput during this 20-workday month was 704 samples/day. Since this purification throughput exceeded the postpurification sample and data handling capabilities provided by commercial solutions, a custom-integrated solution was designed to address these shortcomings. In this paper we detail the key improvements in automation, solvent handling, and sample handling logistics implemented to sustain a mean throughput of 528 samples/day over a multimonth time period.     

Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine

Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.     

Development,validation, and implementation of capillary gel electrophoresis as a replacement for SDS‐PAGE for purity analysis of IgG2 mAbs

Capillary gel electrophoresis (CGE) methods with UV detection were developed for reduced and non‐reduced mAb analysis. These methods can be used to evaluate mAb purity, offering more reproducible quantitation compared with that of traditional SDS‐PAGE methods. These CGE methods have been utilized as platform technology for bioprocess development, formulation development, mAb characterization, drug substance/drug product release testing as well as a required methodology for stability testing. We have found these CGE methods to be applicable across a platform of mAbs in preclinical and clinical development, with the majority of mAbs requiring no modification to the method conditions. This methodology has been ICH validated and transferred to several supporting organizations. The data presented herein describes the development of CGE methodology, platform application to mAb purity analysis, ICH validation, reliability metrics, and considerations on technology enhancement for improved performance and throughput.     

Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns

Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM^®) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM^® Disk with epoxy chemistry. After this, the immobilized CIM^® Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM^® Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap^® metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.     

Efficient Innate Immune Killing of Cancer Cells Triggered by Cell‐Surface Anchoring of Multivalent Antibody‐Recruiting Polymers

Binding of monoclonal antibodies (mAbs) onto a cell surface triggers antibody‐mediated effector killing by innate immune cells through complement activation. As an alternative to mAbs, synthetic systems that can recruit endogenous antibodies from the blood stream to a cancer cell surface could be of great relevance. Herein, we explore antibody‐recruiting polymers (ARPs) as a novel class of immunotherapy. ARPs consist of a cell‐binding motif linked to a polymer that contains multiple small molecule antibody‐binding motifs along its backbone. As a proof of concept, we employ a lipid anchor that inserts into the phospholipid cell membrane and make use of a polymeric activated ester scaffold onto which we substitute dinitrophenol as an antibody‐binding motif. We demonstrate that ARPs allow for high avidity antibody binding and drive antibody recruitment to treated cells for several days. Furthermore, we show that ARP‐treated cancer cells are prone to antibody‐mediated killing through phagocytosis by macrophages.     

The identification of proliferation and tumour-induced proteins in human endothelial cells: a possible target for tumour therapy

The continued growth and spread of tumours is dependent on the proliferation of the endothelial cells of their vasculature. The presence of proliferation- or tumour-induced surface proteins on these endothelial cells would offer a suitable epitope for monoclonal antibody therapy of tumours. Using cultured human umbilical and capillary endothelial cells, we have stimulated them with simple mitogens and tumour conditioned media and examined the proteins induced by [35S]methionine incorporation and 125I-surface-labelling. Two-dimensional polyacrylamide gel electrophoresis revealed the induction of proliferation and tumour-related antigens on the surface of the endothelial cells. Subsequent monoclonal antibody studies suggest that tumour specific surface proteins are present on most tumour endothelium.     

A sub-two minutes method for monoclonal antibody-aggregate quantification using parallel interlaced size exclusion high performance liquid chromatography

In process development and during commercial production of monoclonal antibodies (mAb) the monitoring of aggregate levels is obligatory. The standard assay for mAb aggregate quantification is based on size exclusion chromatography (SEC) performed on a HPLC system. Advantages hereof are high precision and simplicity, however, standard SEC methodology is very time consuming. With an average throughput of usually two samples per hour, it neither fits to high throughput process development (HTPD), nor is it applicable for purification process monitoring. We present a comparison of three different SEC columns for mAb-aggregate quantification addressing throughput, resolution, and reproducibility. A short column (150 mm) with sub-two micron particles was shown to generate high resolution (~1.5) and precision (coefficient of variation (cv)<1) with an assay time below 6 min. This column type was then used to combine interlaced sample injections with parallelization of two columns aiming for an absolute minimal assay time. By doing so, both lag times before and after the peaks of interest were successfully eliminated resulting in an assay time below 2 min. It was demonstrated that determined aggregate levels and precision of the throughput optimized SEC assay were equal to those of a single injection based assay. Hence, the presented methodology of parallel interlaced SEC (PI-SEC) represents a valuable tool addressing HTPD and process monitoring.     

High-throughput profiling of the mitochondrial proteome using affinity fractionation and automation

Recent studies have demonstrated the need for complementing cellular genomic information with specific information on expressed proteins, or proteomics, since the correlation between the two is poor. Typically, proteomic information is gathered by analyzing samples on two-dimensional gels with the subsequent identification of specific proteins of interest by using trypsin digestion and mass spectrometry in a process termed peptide mass fingerprinting. These procedures have, as a rule, been labor-intensive and manual, and therefore of low throughput. The development of automated proteomic technology for processing large numbers of samples simultaneously has made the concept of profiling entire proteomes feasible at last. In this study, we report the initiation of the (eventual) complete profile of the rat mitochondrial proteome by using high-throughput automated equipment in combination with a novel fractionation technique using minispin affinity columns. Using these technologies, approximately one hundred proteins could be identified in several days. In addition, separate profiles of calcium binding proteins, glycoproteins, and hydrophobic or membrane proteins could be generated. Because mitochondrial dysfunction has been implicated in numerous diseases, such as cancer, Alzheimer's disease and diabetes, it is probable that the identification of the majority of mitochondrial proteins will be a beneficial tool for developing drug and diagnostic targets for associated diseases.     

Green metrics for biologics

Biologics are the fastest growing segment of the pharmaceutical market, therefore, the environmental impact of manufacturing these drugs needs to be fully characterized. For monoclonal antibodies, in particular, several metrics have been identified as the manufacturing process is quite standardized for batch processes. This paper will provide an overview of carbon footprint analysis, process mass intensity (PMI), water related impact of energy (WARIEN) and life cycle assessment (LCA) as they have been applied to monoclonal antibody production. Further development and standardization of these tools will allow the industry to identify and implement new technologies that significantly lower the environmental impact of not only monoclonal antibody production but other modalities as well.