Interlaboratory method validation of icIEF methodology for analysis of monoclonal antibodies

CE is central to the analysis, process development and approval of therapeutic monoclonal antibodies (mAbs). Recently, imaged capillary isoelectric focusing (icIEF) has emerged as a powerful technique for quantitative protein charge heterogeneity monitoring and characterization, particularly for mAbs. However, icIEF has yet to be validated for therapeutically relevant mAbs adhering to the ICH guideline (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use). Here, for the first time, icIEF technology was validated by 10 laboratories across 8 independent companies using a therapeutic mAb. The parameters of this method validation strictly follow the guideline of the ICH. This guideline includes specificity, precision, accuracy, linearity, range, LOQ and robustness. These results represent a significant step forward in standardizing the use of icIEF methods for the clinical approval of therapeutic mAbs.     

Rapid multi-attribute characterization of intact bispecific antibodies by a microfluidic chip-based integrated icIEF-MS technology

Rapid, direct identification and quantitation of protein charge variants, and assessment of critical quality attributes with high sensitivity are important drivers required to accelerate the development of biotherapeutics. We describe the use of an enhanced microfluidic chip-based integrated imaged capillary isoelectric focusing-mass spectrometry (icIEF-MS) technology to assess multiple quality attributes of intact antibodies in a single run. Results demonstrate comprehensive detection of multiple charge variants of an aglycosylated knob-into-hole bispecific antibody. Upfront, on-chip separation by icIEF coupled to MS provides the orthogonal separation required to resolve and identify acidic posttranslational modifications including difficult-to-detect deamidation and glycation events at the intact protein level. In addition, on-chip UV detection enables pI determination and relative quantitation of charge isoforms. Six charge variant peaks were resolved by icIEF, mobilized toward the on-chip electrospray tip and directly identified by in-line icIEF-MS using a connected quadrupole time-of-flight mass spectrometer. In addition to acidic charge variants, basic variants were identified as C-terminal lysine, N-terminal cyclization, proline amidation, and the combination of modifications (not typically identified by other intact methods), including lysine and one or two hexose additions. Nonspecific chain cleavages were also resolved, along with their acidic charge variants, demonstrating highly sensitive and comprehensive intact antibody multi-attribute characterization within a 15-min run time.     

Evaluation of an icIEF-MS system for comparable charge variant analysis of biotherapeutics with rapid peak identification by mass spectrometry

Protein therapeutics are usually produced in heterogeneous forms during bioproduction and bioprocessing. Heterogeneity results from post-translational modifications that can yield charge variants and require characterization throughout product development and manufacturing. Isoelectric focusing (IEF) with UV detection is one of the most common methods to evaluate protein charge heterogeneity in the biopharmaceutical industry. To identify charge variant peaks, a new imaged microfluidic chip-based isoelectric focusing (icIEF) system coupled directly to mass spectrometry was recently reported. Bridging is required to demonstrate comparability between existing and new technology. As such, here we demonstrate the comparability of the pI value measurement and relative charge species distributions between the icIEF-MS system and the control data from a frequently utilized methodology in the biopharmaceutical industry for several blinded development-phase biopharmaceutical monoclonal antibodies across a wide pI range of 7.3–9.0. Hyphenation of the icIEF system with mass spectrometry enabled direct and detailed structural determination of a test molecule, with masses suggesting acidic and basic shifts are caused by sialic acid additions and the presence of unprocessed lysine residues. In addition, MS analysis further identified several low-abundance glycoforms. The icIEF-MS system provides sample quantification, characterization, and identification of mAb proteoforms without sacrificing icIEF quantification comparability or speed.     

Applications of imaged capillary isoelectric focussing technique in development of biopharmaceutical glycoprotein-based products

CE-based methods have increasingly been applied to the analysis of a variety of different type proteins. One of those techniques is imaged capillary isoelectric focusing (icIEF), a method that has been used extensively in the field of protein-based drug development as a tool for product identification, stability monitoring, and characterization. It offers many advantages over the traditional labor-intensive IEF slab gel method and even standard cIEF with on-line detection technologies with regard to method development, reproducibility, robustness, and speed. Here, specific examples are provided for biopharmaceutical glycoprotein products such as mAbs, erythropoietin (EPO), and recombinant Fc-fusion proteins, though the technique can be adapted for many other therapeutic proteins. Applications of iCIEF using a Convergent Bioscience instrument (Toronto, Canada) with whole-field imaging technology are presented and discussed. These include a quick method to establish an identity test for many protein-based products, product release, and stability evaluation of glycoproteins with respect to charge heterogeneity under accelerated temperature stress, different pH conditions, and in different formulations. Finally, characterization of glycoproteins using this iCIEF technology is discussed with respect to biosimilar development, clone selection, and antigen binding. The data presented provide a "taste' of what icIEF method can do to support the development of biopharmaceutical glycoprotein products from early clone screening for better product candidates to characterization of the final commercial products.     

A Precise Chemical Strategy To Alter the Receptor Specificity of the Adeno‐Associated Virus

The ability to target the adeno‐associated virus (AAV) to specific types of cells, by altering the cell‐surface receptor it binds, is desirable to generate safe and efficient therapeutic vectors. Chemical attachment of receptor‐targeting agents onto the AAV capsid holds potential to alter its tropism, but is limited by the lack of site specificity of available conjugation strategies. The development of an AAV production platform is reported that enables incorporation of unnatural amino acids (UAAs) into specific sites on the virus capsid. Incorporation of an azido‐UAA enabled site‐specific attachment of a cyclic‐RGD peptide onto the capsid, retargeting the virus to the α_vβ₃ integrin receptors, which are overexpressed in tumor vasculature. Retargeting ability was site‐dependent, underscoring the importance of achieving site‐selective capsid modification. This work provides a general chemical approach to introduce various receptor binding agents onto the AAV capsid with site selectivity to generate optimized vectors with engineered infectivity.     

Analysis of Glycosylated Type II Interleukin-1 Receptor (IL-1R) by Imaged Capillary Isoelectric Focusing (i-cIEF)

Glycoproteins typically produce a complex charge profile due to their heterogeneous glycosylation pattern. We developed a reproducible imaged capillary isoelectric focusing (i-cIEF) method to monitor the charge variants of recombinant human Type II Interleukin-1 receptor (IL-1R), a heavily glycosylated protein expressed as a soluble receptor in Chinese Hamster Ovary (CHO) cells. This method was proved to be informative in multiple settings: monitoring of upstream process and downstream purification, analysis of in-process samples, characterization of the bulk drug substance, as well as testing of stability samples during drug development. The i-cIEF method was efficient at detecting changes in the charge isoform profile during different steps of the purification procedures or resulting from modifications of cell culture conditions. In addition, this method was well suited to monitor the consistency of sialic acid distribution and to detect deamidation events occurring in accelerated stability studies. The i-cIEF method presented here provides an improvement over IEF slab gels in terms of resolution, automation and quantitation. Due to its exquisite resolution of narrow pI range, this technology can find applications in quality control environment as identity assay as well as in the analytical laboratory to monitor subtle modifications of the protein.

     

Analysis of Glycosylated Type II Interleukin-1 Receptor (IL-1R) by Imaged Capillary Isoelectric Focusing (i-cIEF)

Glycoproteins typically produce a complex charge profile due to their heterogeneous glycosylation pattern. We developed a reproducible imaged capillary isoelectric focusing (i-cIEF) method to monitor the charge variants of recombinant human Type II Interleukin-1 receptor (IL-1R), a heavily glycosylated protein expressed as a soluble receptor in Chinese Hamster Ovary (CHO) cells. This method was proved to be informative in multiple settings: monitoring of upstream process and downstream purification, analysis of in-process samples, characterization of the bulk drug substance, as well as testing of stability samples during drug development. The i-cIEF method was efficient at detecting changes in the charge isoform profile during different steps of the purification procedures or resulting from modifications of cell culture conditions. In addition, this method was well suited to monitor the consistency of sialic acid distribution and to detect deamidation events occurring in accelerated stability studies. The i-cIEF method presented here provides an improvement over IEF slab gels in terms of resolution, automation and quantitation. Due to its exquisite resolution of narrow pI range, this technology can find applications in quality control environment as identity assay as well as in the analytical laboratory to monitor subtle modifications of the protein.     

A high-resolution capillary isoelectric focusing method for the determination of therapeutic recombinant monoclonal antibody

Capillary isoelectric focusing (CIEF) is a common choice for separation and analysis of the charge variants and impurities of therapeutic proteins. In this study, we developed a sensitive CIEF analysis method for determining the charge heterogeneity of therapeutic monoclonal antibody (mAb) using Beckman PA800 plus platform. The mixture of 5% Pharmalyte 8-10.5 and 1% Pharmalyte 3-10 was used to overcome the limitation of using single Pharmalyte 3-10 in detecting charge heterogeneity of basic mAb. This approach largely improved the resolution of the heterogeneous peaks. In addition, 3 M urea and 50 mM arginine (Arg) were used to improve the separation as solubilizer and cathodic stabilizer, respectively. Under optimized condition, both acidic and basic peaks of the mAb were separated well. Method qualification results showed good specificity, precision, and linearity within the concentration range of 0.03-0.20 mg/mL for mAb R1. The method was then used for C-terminal lysine (Lys) variants characterization and glycosylation profiles analysis. Furthermore, it also had a wide application in the clone screening process. The highly sensitive and repeatable results highlighted the wide application prospects of this method in biopharmaceutical industry.     

Microheterogeneity of human erythrocyte pyruvate kinase: application of immunological visualization techniques after isoelectric focusing in ultrathin gels

Immunological methods for protein visualization afford high sensitivity, good specificity and resolution. We used the indirect immunoassay with peroxidase- and 125I-labelled antibodies to investigate the microheterogeneity of pyruvate kinase from human red blood cells and can show an enzyme pattern with more than 10 single bands. Different methods were tested as to their applicability for protein fixation and subsequent immunological visualization in polyacrylamide gels. Besides the Western blot, immunofixation and ethanolic fixation can be recommended as fixation techniques, especially after isoelectric focusing in ultrathin gels.     

Enzyme visualization with partially dehydrated agarose substrate layers: detection of paraoxonase after thin-layer isoelectric focusing in agarose

A new approach is described for studying the polymorphism of paraoxon hydrolyzing serum esterases after isoelectric focusing of native sera. Enzyme visualization is performed by a modified sandwich procedure which is faster and also affords higher resolution and considerably improved sensitivity. Up to seven paraoxon splitting isoenzymes can be visualized and clearly distinguished from arylesterases and phosphatases by using 3-naphtyl acetate, paraoxon, 5-bromo-4-chloro-3-indolyl acetate and 5-bromo-4-chloro-3-indolyl phosphate as substrates. The new technique is also able to differentiate between paraoxonase isoenzymes sensitive to EDTA and those which are EDTA-stable. Immunofixation with anti-human serum albumin-antibodies revealed similar isoelectric points for these isoenzymes, although they are not assumed to be identical. The new technique may prove useful in other applications of enzyme visualization where diffusion of enzymes and/or cleavage products is the major problem.     

Immobilization of E. coli with autodisplayed Z-domains to a surface-modified microplate for immunoassay

Escherichia coli with autodisplayed Z-domains was reported to improve the sensitivity of immunoassays by the orientation control of antibodies. In this work, a sensitive microplate-based immunoassay is presented by immobilizing E. coli cells to a surface-modified microplate. The microplate was prepared by coating parylene-H film with formyl groups, and then covalently coupling poly-L-lysine to the parylene-H film. The E. coli cells were bound to the microplate by charge interactions between the negatively charged E. coli outer membrane and the positively charged microplate surface. In this work, the preparation of the microplate coated with poly-L-lysine is presented. The immobilization efficiency of E. coli to the modified surface was estimated to be far higher than non-specific interaction by fluorescence microscope and the optical transmittance of the modified microplate was measured to be feasible for immunoassay. The microplate-based immunoassay is demonstrated to be feasible for medical diagnosis of inflammatory diseases by using C-reactive protein as a target analyte for the medical diagnosis of inflammatory diseases.     

Characterization of each isoform of a F(ab')2 by capillary electrophoresis.

Free solution capillary electrophoresis was investigated for the characterization of an M(r) 100 000 purified F(ab')2. Optimization of the experimental conditions allowed the identification of five separated peaks, suggesting the presence of isoforms which differed by only 0.2 pH unit. This heterogeneity was still detectable with 80 amol of protein. After a preparative separation by chromatofocusing, identification of each form was performed for the first time by capillary electrophoresis. A quantitative and qualitative correlation with isoelectric focusing showed that free solution capillary electrophoresis represents a sensitive method for revealing subtle differences in charge, even for large proteins.     

A novel piezoelectric immunosensor for detection of carcinoembryonic antigen

A simple, rapid, and highly sensitive immunosensor for the direct determination of carcinoembryonic antigen (CEA) in human serum using a piezoelectric crystal has been developed and optimized. In order to improve sensitivity of the immunosensor, a protein A-based orientation-controlled immobilization method for antibodies was adopted together with an immunoreactive accelerant of polyethyleneglycol (PEG) used to amplify the signal response of frequency. Human normal serum was utilized as a reference background. The linear range for CEA concentration obtained by the end-point method was 66.7-466.7 ng/mL. Clinical samples from cancer patients were analyzed by the proposed piezoelectric immunoassay, and the analytical results were reasonably comparable with those obtained by the chemiluminescence immunoassay (CLIA). The proposed immunosensor provides a new promising method for the highly sensitive immunoassay of CEA in clinical laboratory.     

Charge heterogeneity of a therapeutic monoclonal antibody conjugated with a cytotoxic antitumor antibiotic, calicheamicin

A robust and highly reproducible capillary isoelectric focusing (cIEF) method for the evaluation of charge heterogeneity of monoclonal antibody (mAb) pharmaceutical which contains covalently bound antitumor compounds was developed using a combination of commercially available dimethylpolysiloxane-coated capillary and carrier ampholyte. In order to optimize major analytical parameters for robust mobilization, experimental responses from three pI markers were selected. The optimized method gave excellent repeatability and intermediate precision in estimated pI values of charge variants with relative standard deviations (RSDs) of not more than 0.06% and 0.95%, respectively, when using IgG(4) as a model. Furthermore, RSDs of charge variant compositions were less than 5.0%. These results suggest that the proposed method can be a powerful tool for reproducible evaluation of charge variants of both naked mAbs and their conjugates with high resolution, and it is applicable to quality testing and detailed characterization in the pharmaceutical industry. In addition, it should be noticed that the method provided non-linear pH gradient within the tested ranges, from pI 9.50 to 3.78, and the pH gradient caused the inconsistency of estimated pI ranges between cIEF and gel IEF. This result indicates that selecting appropriate pI markers based on the target pI ranges of charge variants for each mAb related pharmaceutical is highly recommended for the precise determination of pI values.     

SERS-based immunoassay using a gold array-embedded gradient microfluidic chip

Here we report the development of a programmable and fully automatic gold array-embedded gradient microfluidic chip that integrates a gradient microfluidic device with gold-patterned microarray wells. This device provides a convenient and reproducible surface-enhanced Raman scattering (SERS)-based immunoassay platform for cancer biomarkers. We used hollow gold nanospheres (HGNs) as SERS agents because of their highly sensitive and reproducible characteristics. The utility of this platform was demonstrated by the quantitative immunoassay of alpha-fetoprotein (AFP) model protein marker. Our proposed SERS-based immunoassay platform has many advantages over other previously reported SERS immunoassay methods. The tedious manual dilution process of repetitive pipetting and inaccurate dilution is eliminated with this process because various concentrations of biomarker are automatically generated by microfluidic gradient generators with N cascade-mixing stages. The total assay time from serial dilution to SERS detection takes less than 60 min because all of the experimental conditions for the formation and detection of immunocomplexes can be automatically controlled inside the exquisitely designed microfluidic channel. Thus, this novel SERS-based microfluidic assay technique is expected to be a powerful clinical tool for fast and sensitive cancer marker detection.     

基于水溶性共轭聚合物分子刷的肿瘤标志物快速检测

以水溶性聚对苯撑乙炔分子刷(PPEB)为传感材料,荧光素标记的多肽为识别探针,设计了一种可实现肿瘤标志物快速、灵敏检测的蛋白质传感器。PPEB的刷状结构带有大量正电荷,与带负电荷的多肽形成静电复合物,使能量供体(PPEB)与受体(荧光素)之间的距离较近,发生高效荧光共振能量转移(FRET)。当多肽探针被前列腺特异性抗原(PSA)识别并切割为更小的寡肽片段时,由于肽段所带电荷量和等电点的变化,其与PPEB之间的静电作用减弱,能量供体和受体之间的距离增大,FRET效率降低。该传感器对PSA的检测可在40min内完成,具有良好的特异性和灵敏度。     

Size‐matched alkyne‐conjugated cyanine fluorophores to identify differences in protein glycosylation

Currently, there are few methods to detect differences in posttranslational modifications (PTMs) in a specific manner from complex mixtures. Thus, we developed an approach that combines the sensitivity and specificity of click chemistry with the resolution capabilities of 2D‐DIGE. In “Click‐DIGE”, posttranslationally modified proteins are metabolically labeled with azido‐substrate analogs, then size‐ and charge‐matched alkyne‐Cy3 or alkyne‐Cy5 dyes are covalently attached to the azide of the PTM by click chemistry. The fluorescently‐tagged protein samples are then multiplexed for 2DE analysis. Whereas standard DIGE labels all proteins, Click‐DIGE focuses the analysis of protein differences to a targeted subset of posttranslationally modified proteins within a complex sample (i.e. specific labeling and analysis of azido glycoproteins within a cell lysate). Our data indicate that (i) Click‐DIGE specifically labels azido proteins, (ii) the resulting Cy‐protein conjugates are spectrally distinct, and (iii) the conjugates are size‐ and charge‐matched at the level of 2DE. We demonstrate the utility of this approach by detecting multiple differentially expressed glycoproteins between a mutant cell line defective in UDP‐galactose transport and the parental cell line. We anticipate that the diversity of azido substrates already available will enable Click‐DIGE to be compatible with analysis of a wide range of PTMs.     

Design and synthesis of new fluorescent probe for rapid and highly sensitive detection of proteins via electrophoretic gel stain

A new fluorescent molecular probe, 2,2′‐(1E,1′E)‐2,2′‐(4‐(dicyanomethylene)‐4H‐pyrane‐2,6‐diyl)bis(ethene‐2,1‐diyl)bis(sodium benzenesulfonate) salt ( 1 ), possessing the cyanopyranyl moieties and two benzene sulfonic acid groups was designed and synthesized to detect proteins in solution and for high‐throughput SDS‐PAGE. Compound 1 exhibited no fluorescence in the absence of proteins; however, it exhibited strong fluorescence on the addition of bovine serum albumin as a result of intramolecular charge transfer. Compared with the conventional protocols for in‐gel protein staining, such as SYPRO Ruby and silver staining, 1 achieves higher sensitivity, even though it offers a simplified, higher throughput protocol. In fact, the total time required for protein staining was 60–90 min under optimum conditions much shorter than that required by the less‐sensitive silver staining or SYPRO Ruby staining protocols. Moreover, 1 was successfully applied to protein identification by mass spectrometry via in‐gel tryptic digestion, Western blotting, and native PAGE together with protein staining by 1 , which is a modified protocol of blue native PAGE (BN‐PAGE). Thus, 1 may facilitate high‐sensitivity protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.     

Charge heterogeneity of recombinant pro-urokinase and urinary urokinase, as revealed by isoelectric focusing in immobilized pH gradients

When analysing homogeneous preparations of recombinant pro-urokinase and urinary urokinase by isoelectric focusing (IEF) in immobilized pH gradients, an extreme charge heterogeneity was detected (at least ten major and ten minor bands in the pH range 7–10). This extensive polydispersity was not caused by different degrees of glycosylation, or by IEF artefacts, such as binding to carrier ampholytes or carbamylation by urea. A great part of this heterogeneity could be traced back to the existence of a multitude of protein molecules containing Cys residues at different oxidation levels (-SH, -S-S-, even cysteic acid). Owing to the very large number of Cys residues in pro-urokinase (24 out of a total of 411 amino acids) and to the relatively high pI of its native forms (pI 9.5–9.8; the native form is believed to contain all Cys residues as -S-S- bridges), the presence of SH or cysteic acid residues would increase the negative surface charge, as even SH groups would be extensively ionized. In pro-urokinase, part of the heterogeneity was also due to spontaneous degradation to urokinase and possibly also to cleavage into lower-molecular-mass fragments. When all these causes of heterogeneity were removed, the pI spectrum was reduced to only four, about equally intense, bands. The cause of this residual heterogeneity is unknown.