Combined immuno-purification and detection of recombinant erythropoietins and activin receptor type II-Fc fusion proteins by isoelectric focusing for application in doping control.

Iso-electric focusing (IEF) was the first method established to discriminate endogenous and recombinant erythropoietins (rEPOs). It is still approved by the World Anti-Doping Agency (WADA) as an initial testing procedure to detect erythropoiesis stimulating agents (ESAs) in doping control samples. However EPO-Fc, one of the prohibited rEPOs designated by WADA, is not detectable with the actual IEF conditions. Other newly developed ESAs - luspatercept and sotatercept, both activin receptor type II-Fc fusion proteins (ActRII-Fc) - are also now prohibited and could be used in combination with rEPOs. Methods of identification of ActRII-Fc in blood by SAR/SDS-PAGE have been described, but not by IEF. Here we detail improvements in blood sample preparation and IEF analysis: A combined immuno-purification of EPOs and ActRII-Fc proteins in a single procedure, an appropriate isoforms separation for all proteins using new pre-loading and gel conditions, and a single detection of all rEPOs and ActRII-Fc proteins after successive incubation with anti-EPO and anti-ActRII antibodies. With these changes, distinctive profiles for all the ESAs were obtained by IEF. Therefore, IEF could be used as a screening method to detect a wide spectrum of prohibited ESAs in blood samples prior to specific confirmation for the identified rEPO or ActRII-Fc.

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.

Qualification of NISTmAb charge heterogeneity control assays.

The NISTmAb is a monoclonal antibody Reference Material from the National Institute of Standards and Technology; it is a class-representative IgG1κ intended serve as a pre-competitive platform for harmonization and technology development in the biopharmaceutical industry. The publication series of which this paper is a part describes NIST's overall control strategy to ensure NISTmAb quality and availability over its lifecycle. In this paper, the development and qualification of methods for monitoring NISTmAb charge heterogeneity are described. Capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF) assays were optimized and evaluated as candidate assays for NISTmAb quality control. CIEF was found to be suitable as a structural characterization assay yielding information on the apparent pI of the NISTmAb. CZE was found to be better suited for routine monitoring of NISTmAb charge heterogeneity and was qualified for this purpose. This paper is intended to provide relevant details of NIST's charge heterogeneity control strategy to facilitate implementation of the NISTmAb as a test molecule in the end user's laboratory. Graphical Abstract Representative capillary zone electropherogram of the NIST monoclonal antibody (NISTmAb). The NISTmAb is a publicly available research tool intended to facilitate advancement of biopharmaceutical analytics.

Microscale isolation of native forms of lysozyme from chicken egg white by gel isoelectric focusing.

To separate and extract the native states of lysozyme from chicken egg white, a hybrid method for the mobilization of proteins after non-denaturing gel isoelectric focusing (IEF) combined with detection of lysozyme activity was developed. When the proteins in the chicken egg white were first separated using non-denaturing gel IEF, a lysozyme was obtained at the top of the gel column at the cathode end of the IEF. And, when the IEF-separated proteins of the egg white were mobilized by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, an additional active state of the lysozyme that could be bound to proteins, such as ovotransferrin, was extracted from the solution. Furthermore, it was shown that the addition of lysozyme, obtained via IEF, to pure ovotransferrin generated a complex manifesting lysozyme activity, clearly indicating a successful reconstruction of the lysozyme-ovotransferrin complex in vitro. Therefore, the obtained results demonstrated that the native states of lysozymes, such as lysozyme and the lysozyme-ovotransferrin complex, can be effectively separated and extracted using non-denaturing gel IEF. Thus, this method can be applied to separate and extract different charge states of native proteins that retain their biological activities.

Fc-fragment removal allows the EPO-Fc fusion protein to be detected in blood samples by IEF-PAGE.

EPO-Fc proteins have been under investigation as a potential drug for treating anaemia and have shown larger half-life values than other erythropoiesis-stimulating agents (ESAs). Sodium dodecyl sulfate/sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SDS/SAR-PAGE) methods and subsequent immunoblotting are used for routine anti-doping analysis. This paper reports that EPO-Fc fusion proteins can be detected in serum samples by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) in carrier ampholyte-based gels with a pH 2-6 gradient after removing the Fc part via site-specific IdeS protease cleavage. The IdeS-digested EPO-Fc protein yields three fragments: two Fc fragments and one dimeric EPO-hinge fragment. After IEF-PAGE was followed by double Western blotting with chemiluminescent detection, the dimeric EPO-hinge fragment showed a unique isoelectric pattern, which differed from those of any other currently known analogue of EPO. We observed that the removal of the Fc fragment from EPO-Fc reduced the apparent molecular weight of entire fusion protein and increased its electrophoretic mobility. As a result, the band for the EPO-hinge fragment was located in a region between the rEPO and NESP standards, at which lower amounts of serum proteins are present. Simple and selective protocols for determining the EPO-Fc protein in human serum were developed to extend the methodological anti-doping arsenal. This protocol has been characterized. The limit of detection (LOD) of the IEF-PAGE method was 20 pg, and that of SDS/SAR-PAGE was 15 pg.

Application of capillary electrophoresis in glycoprotein analysis.

Capillary electrophoresis (CE) is a versatile analytical method used to characterize glycoproteins. We have used several modes of CE separation such as CE-SDS gel, imaged capillary isoelectric focusing (icIEF), and capillary zone electrophoresis (CZE) to study therapeutic glycoprotein products. CE-SDS gel is applied to characterize the glycan occupancy and number of glycosylation sites, and icIEF is used to study the charge heterogeneities due to sialic acids in glycoproteins. To further characterize the glycoprotein, removal of N-linked glycans is necessary and a CZE technique is employed to analyze each glycan moiety. Examples from a monoclonal antibody, erythropoietin, and granulocyte colony-stimulating factor are presented here to demonstrate the utility of these CE modes. The details of sample preparation and separation conditions for each CE mode are described in this chapter.

CIEF method optimization: development of robust and reproducible protein reagent characterization in the clinical immunodiagnostic industry.

Several method parameters have been refined for application of CIEF methods to provide optimal capillary robustness and performance longevity while maintaining desired analytical output for the ever increasing characterization scrutiny of protein reagents used in clinical assay formulations. Demonstrated here are significant modifications to the existing protocols in order to attain a robust, reproducible method that achieves as much as a 20-fold increase in the number of consecutive runs before capillary degradation. Not only is it a concern for the rudimentary analysis of acidic and basic components of the isoform profile for monoclonal antibodies, but a comprehensive identification of each individual isoform to obtain a characteristic fingerprint is necessary for minor distinguishable properties between multiple proteins in unambiguous identification. In order to maintain the integrity of these modifications, extensive studies were conducted on an implemented system suitability standard protein with specifically defined parameters indicating either sufficient or poor separation performance.

Robustness of iCIEF methodology for the analysis of monoclonal antibodies: an interlaboratory study.

An international team including 12 laboratories from 11 independent biopharmaceutical companies in the United States and Switzerland was formed to evaluate the precision and robustness of imaged capillary isoelectric focusing for the charge heterogeneity analysis of monoclonal antibodies. The different laboratories determined the apparent pI and the relative distribution of the charged isoforms for a representative monoclonal antibody sample using the same capillary isoelectric focusing assay. Statistical evaluation of the data was performed to determine within and between laboratory consistencies and outlying information. The apparent pI data generated for each charged variant peak showed very good precision between laboratories with RSD values of less than 0.8%. Similarly, the RSD for the therapeutic monoclonal antibody charged variants percent peak area values are less than 11% across different laboratories using different analyst, different lots of ampholytes and multiple instruments. These results validate the appropriate use of imaged capillary isoelectric focusing in the biopharmaceutical industry in support of process development and regulatory submissions of therapeutic antibodies.

Immunofixation electrophoresis for identification of proteins and specific antibodies.

Immunofixation electrophoresis (IFE) is a technique for the identification of proteins within complex mixtures after separation by either conventional zone electrophoresis or isoelectric focusing. Most commonly antigens (which are often immunoglobulins) are separated by electrophoresis followed by precipitation with specific antibodies in situ. However, immunoglobulins with specific reactivity can be also precipitated with the proper antigens after electrophoresis in reverse or reversed IFE. Because of its great versatility, potentially high sensitivity, ease to perform and customize, and relatively low cost with no requirement for expensive instrumentation, manual IFE remains a valuable tool for both clinical diagnostic testing and research. Any low-viscosity body fluid specimen or, possibly, culture fluid could be tested with IFE if proper antibodies (or antigens in reverse[d] IFE) are available. After pretreatment with chaotropic and/or reducing agents, even high-viscosity specimens might be amenable to testing with IFE.

Two-dimensional gel electrophoresis: vertical isoelectric focusing.

Two-dimensional gel electrophoresis (2-DE) is one of the most powerful tools for separating proteins based on their size and charge. 2-DE is very useful to separate two proteins with identical molecular weights but different charges, which cannot be achieved with just sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Here, a simpler and easier version of 2-DE is presented which is also faster than all the currently available techniques. In this modified version of 2-DE, isoelectric focusing is carried out in the first dimension using a vertical SDS-PAGE apparatus. Following the first-dimensional IEF, each individual lane is excised from the IEF gel and, after a 90° rotation, is inserted into a second-dimensional SDS-PAGE, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This version of IEF can be run in less than 2 h compared to the overnight run required by O'Farrell's method. Difficult tube gel casting and gel extrusion as well as tube gel distortion are eliminated in our method. This method is simpler, faster, and inexpensive. Both dimensions can be done on the same SDS-PAGE apparatus, and up to ten samples can be run simultaneously using one gel.

Isoelectric focusing in agarose gel for detection of oligoclonal bands in cerebrospinal and other biological fluids.

Isoelectric focusing (IEF) coupled with immunodetection (immunofixation or immunoblotting) has become the leading technique for the detection and study of oligoclonal bands (OCBs) in cerebrospinal fluid (CSF) and also is increasingly used in other body fluids such as the tear and serum. Limited commercial availability of precast agarose IEF gels for research and a need for customization prompted reporting a detailed general protocol for the preparation and casting of agarose IEF gel along with sample, control, and isoelectric point marker preparation and carrying out the focusing itself for CSF OCBs. However, the method is readily adaptable to the use of other body fluid specimens and, possibly, research specimens such as culture fluids as well.

Isobaric labeling and data normalization without requiring protein quantitation.

Isobaric multiplexed quantitative proteomics can complement high-resolution sample isolation techniques. Here, we report a simple workflow exponentially modified protein abundance index (emPAI)-MW deconvolution (EMMOL) for normalizing isobaric reporter ratios within and between experiments, where small or unknown amounts of protein are used. EMMOL deconvolutes the isobaric tags for relative and absolute quantification (iTRAQ) data to yield the quantity of each protein of each sample in the pool, a new approach that enables the comparison of many samples without including a channel of reference standard. Moreover, EMMOL allows using a sufficient quantity of control sample to facilitate the peptide fractionation (isoelectric-focusing was used in this report), and mass spectrometry MS/MS sequencing yet relies on the broad dynamic range of iTRAQ quantitation to compare relative protein abundance. We demonstrated EMMOL by comparing four pooled samples with 20-fold range differences in protein abundance and performed data normalization without using prior knowledge of the amounts of proteins in each sample, simulating an iTRAQ experiment without protein quantitation prior to labeling. We used emPAI, the target protein MW, and the iTRAQ reporter ratios to calculate the amount of each protein in each of the four channels. Importantly, the EMMOL-delineated proteomes from separate iTRAQ experiments can be assorted for comparison without using a reference sample. We observed no compression of expression in iTRAQ ratios over a 20-fold range for all protein abundances. To complement this ability to analyze minute samples, we report an optimized iTRAQ labeling protocol for using 5 µg protein as the starting material.

Cave digitabula!

We report here observations clearly demonstrating that gloves can have a deleterious impact on an IEF experiment. These observations were made during the practice of analyses for anti-doping control of erythropoietin. We show that the wearing of nitrile gloves to apply the catholyte strip onto the IEF gel may be responsible for dramatic distortions in the pattern of this hormone. These observations point out that gloves must not be considered only as protective items but also as possible factors in an analytic process.

Isoelectric focusing management: an investigation for salt interference and an algorithm for optimization.

Two-dimensional electrophoresis (2-DE) has evolved into a robust separation technique in proteomic research. However, one of the major challenges in 2-DE experiments, the reproducibility of the first dimensional electrophoresis (IEF), has remained unsolved. It is well-known that the quality of IEF experiments is significantly affected by the salt interference. Nevertheless, the interference mechanisms of salts in IEF have never been systematically investigated. In this study, we comprehensively investigated the interference effects in IEF due to various kinds of simple and buffer salts in protein samples. Two interference schemes were proposed accordingly to elucidate the interference mechanisms of salts in IEF. Furthermore, to increase the reproducibility of IEF, we proposed that conductivity measurement is a feasible method to assess the salt content of 2-DE samples and developed an algorithm to predict the optimal total volt-hours (Vh) required for protein focusing in IEF. The developed algorithm had been evaluated under various IEF conditions for a variety of 2-DE samples and proven to be a reliable guide. In sum, information disclosed in this study should be of use for increasing the reproducibility and thus the applicability of 2-DE in current proteomics.

Procedures for monitoring recombinant erythropoietin and analogs in doping.

Hemoglobin concentration is one of the principal factors of aerobic power and, consequently, of performance in many types of physical activities. The use of recombinant human erythropoietin is, therefore, particularly powerful for improving the physical performances of patients, and, more generally, improving their quality of life. This article discusses procedures for monitoring recombinant erythropoietin and its analogues in doping for athletic performance.

Evaluation of a new Sebia isoelectrofocusing kit for alpha 1-antitrypsin phenotyping with the Hydrasys System.

BACKGROUND: Laboratory evaluation of alpha 1-antitrypsin (A1AT) deficiency is generally performed by determination of A1AT concentrations and identification of specific allelic variants by phenotyping. For this purpose, we evaluated a new Hydragel 18 A1AT Isofocusing kit on the semi-automatic Hydrasys System (Sebia) for the determination of A1AT phenotypes by isoelectrofocusing on ready-to-use agarose gels with specific immunological detection. METHODS: Serum samples from 66 patients were analysed with this new kit in comparison with the conventional and manually performed isoelectrofocusing method on polyacrylamide gels with Coomassie Blue staining. RESULTS: A1AT phenotypes showed comparable iso-electrofocusing patterns in both systems. The good within-gel reproducibility of this kit was demonstrated using two normal serum samples (M1 and M1M2 phenotypes) and six pathological serum samples with different phenotypes (MS, SS, SZ, MZ, ZZ). A sensitivity study was undertaken by performing serial dilutions on a serum with a ZZ phenotype containing 0.27 g/L A1AT. The detection limit was 0.050 g/L. CONCLUSIONS: This new method is highly specific, rapid and simple to perform. It improves identification of not only the most common but also various rare A1AT phenotypes. It appears to be suitable for routine analysis and screening applications in a clinical laboratory setting.

Cascaded free-flow isoelectric focusing for improved focusing speed and resolution.

This work presents the first implementation of cascaded stages for a microfabricated free-flow isoelectric focusing (FF-IEF) device. Both analytical and computational models for IEF suggest device performance will be improved by utilizing multiple stages to reduce device residence time. These models are shown to be applicable by using focusing of small IEF markers as a demonstration. We also show focusing of fluorescently tagged proteins under different channel geometries, with the most efficient focusing occurring in the cascaded design, as predicted by theory. An additional aim of this work is to demonstrate the compatibility of cascaded FF-IEF with common bioanalytical tools. As an example, outlet fractions from cascaded FF-IEF were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Processing of whole cell lysate followed by immunoblotting for cell signaling markers demonstrates the reduction of albumin from samples, as well as the enrichment of apoptotic markers.

Oligoclonal bands in multiple sclerosis cerebrospinal fluid: an update on methodology and clinical usefulness.

Two or more oligoclonal IgG bands (OB) detected by separation of cerebrospinal fluid (CSF) proteins while not demonstrable in corresponding serum reflect a local B-cell response accompanying central nervous system (CNS) inflammation. Using optimized, standardized methodology, preferentially protein separation by isoelectric focusing followed by immunoblotting, more than 95% of patients with multiple sclerosis (MS) have CSF OB of IgG class not detectable in serum, thereby providing powerful evidence for the diagnosis of MS. Once present, CSF OB persists in the individual patient irrespective of MS course or therapy. Because of the high sensitivity of CSF OB in MS as well as its high specificity in the appropriate clinical setting, examination of CSF for OB of IgG class can be strongly recommended to obtain support for the diagnosis of MS and identify patients with clinically isolated syndrome (CIS) at increased risk of developing MS. The IgG index equal to CSF/serum IgG:CSF/serum albumin is elevated in about 70% of MS patients, but rarely in CSF OB-negative MS. Because of lower diagnostic sensitivity, IgG index cannot be recommended as replacement of CSF OB in the diagnosis of MS but, when elevated, as additional evidence for an augmented B-cell response within the CNS that is compatible with MS. Although the clinical picture as well as findings from magnetic resonance imaging of the brain and spinal cord are essential for an MS diagnosis, this should be re-evaluated in CSF OB-negative patients, keeping in mind the many disease entities imitating MS. Recommended diagnostic criteria for MS must include definitions of the role of lumbar puncture and of clearly specified, optimized and standardized routine CSF investigations including for the presence of CSF IgG OB. There is a need for concerted long-term follow-up studies of the subgroup of MS patients without CSF OB regarding e.g. prognostic and immunologic features. For inclusion in trials of disease-modulating drugs, it is recommended that patients with MS or CIS are selected regarding presence vs. absence of CSF OB. Development and evaluation of new technologies to define local vs. systemic B-cell responses in patients with MS or CIS vs. patients with other inflammatory neurological diseases should shed new light on the role of CSF OB, which remains enigmatic.