Possibility of using the imaged capillary isoelectric method as a multi-attribute method for bispecific antibodies.

An imaged capillary isoelectric focusing (icIEF)-based method was developed and validated as a multi-attribute method for a bispecific antibody (BsAb). First, as the traditional application of the icIEF method, it serves as an identity assay and purity assay for the BsAb. Second, the method can also be used as an impurity assay for the homodimer monoclonal antibodies generated during BsAb assembly. The homodimer impurity analysis for BsAb is usually done by hydrophobic interaction chromatography methods in the industry. The icIEF method has good sensitivity (down to 4 µg/mL in a limit of quantitation) when UV fluorescence detection is used, which detects the native fluorescence of proteins. This is the first report that an icIEF method has been applied as impurity assay.

Microstrip isoelectric focusing with deep learning for simultaneous screening of diabetes, anemia, and thalassemia.

BACKGROUND: Hemoglobin (Hb) is an important protein in red blood cells and a crucial diagnostic indicator of diseases, e.g., diabetes, thalassemia, and anemia. However, there is a rare report on methods for the simultaneous screening of diabetes, anemia, and thalassemia. Isoelectric focusing (IEF) is a common separative tool for the separation and analysis of Hb. However, the current analysis of IEF images is time-consuming and cannot be used for simultaneous screening. Therefore, an artificial intelligence (AI) of IEF image recognition is desirable for accurate, sensitive, and low-cost screening. RESULTS: Herein, we proposed a novel comprehensive method based on microstrip isoelectric focusing (mIEF) for detecting the relative content of Hb species. There was a good coincidence between the quantitation of Hb via a conventional automated hematology analyzer and the one via mIEF with R2 = 0.9898. Nevertheless, our results showed that the accuracy of disease diagnosis based on the quantification of Hb species alone is as low as 69.33 %, especially for the simultaneous screening of multiple diseases of diabetes, anemia, alpha-thalassemia, and beta-thalassemia. Therefore, we introduced a ResNet1D-based diagnosis model for the improvement of screening accuracy of multiple diseases. The results showed that the proposed model could achieve a high accuracy of more than 90 % and a good sensitivity of more than 96 % for each disease, indicating the overwhelming advantage of the mIEF method combined with deep learning in contrast to the pure mIEF method. SIGNIFICANCE: Overall, the presented method of mIEF with deep learning enabled, for the first time, the absolute quantitative detection of Hb, relative quantitation of Hb species, and simultaneous screening of diabetes, anemia, alpha-thalassemia, and beta-thalassemia. The AI-based diagnosis assistant system combined with mIEF, we believe, will help doctors and specialists perform fast and precise disease screening in the future.

Two-Dimensional Gel Electrophoresis for Protein Separation of Plant Samples.

This chapter provides a description of the procedure for two-dimensional electrophoresis that can be performed for any given gel size and isoelectric focusing range. This will enable the operator to recognize critical steps and gain sufficient information to generate 2D images suitable for computer-assisted analysis of 2D-gel, as well as mass spectrometry analysis for protein identification and characterization.

"Negative" Impact: The Role of Payload Charge in the Physicochemical Stability of Auristatin Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) tend to be less stable than their parent antibodies, which is often attributed to the hydrophobic nature of their drug payloads. This study investigated how the payload charge affects ADC stability by comparing two interchain cysteine ADCs that had matched drug-to-antibody ratios and identical linkers but differently charged auristatin payloads, vcMMAE (neutral) and vcMMAF (negative). Both ADCs exhibited higher aggregation than their parent antibody under shaking stress and thermal stress conditions. However, conjugation with vcMMAF increased the aggregation rates to a greater extent than conjugation with uncharged but more hydrophobic vcMMAE. Consistent with the payload logD values, ADC-vcMMAE showed the greatest increase in hydrophobicity but minor changes in charge compared with the parent antibody, as indicated by hydrophobic interaction chromatography and capillary electrophoresis data. In contrast, ADC-vcMMAF showed a decrease in net charge and isoelectric point along with an increase in charge heterogeneity. This charge alteration likely contributed to a reduced electrostatic repulsion and increased surface activity in ADC-vcMMAF, thus affecting its aggregation propensity. These findings suggest that not only the hydrophobicity of the payload, but also its charge should be considered as a critical factor affecting the stability of ADCs.

Mass spectrometry study on SARS-CoV-2 recombinant vaccine with comprehensive separation techniques to characterize complex heterogeneity.

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation and recombinant vaccine has gained much attention thanks to its potential for greater response predictability, improved efficacy, rapid development and reduced side effects. Recombinant vaccines are designed and manufactured using bacterial, yeast cells or mammalian cells. A small piece of DNA is taken from the virus or bacterium against which we want to protect and inserted into the manufacturing cells. Due to the extremely complex heterogeneity of SARS-CoV-2 recombinant vaccine, single technology platform cannot achieve thorough and accurate characterization of such difficult proteins so integrating comprehensive technologies is essential. This study illustrates an innovative workflow employing multiple separation techniques tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of SARS-CoV-2 recombinant vaccine, including ultra-high performance liquid chromatography (UHPLC), ion exchange chromatography (IEX) and imaged capillary isoelectric focusing (icIEF). The integrated methodology focuses on the importance of cutting-edge icIEF-MS online coupling and icIEF fractionation applied to revealing the heterogeneity secret of SARS-CoV-2 recombinant vaccine.

Identification of structural origins of complex charge heterogeneity in therapeutic ACE2Fc fusion protein facilitated by free-flow isoelectric focusing.

Fc Fusion protein represents a versatile molecular platform with considerable potential as protein therapeutics of which the charge heterogeneity should be well characterized according to regulatory guidelines. Angiotensin-converting enzyme 2 Fc fusion protein (ACE2Fc) has been investigated as a potential neutralizing agent to various coronaviruses, including the lingering SARS-CoV-2, as this coronavirus must bind to ACE2 to allow for its entry into host cells. ACE2Fc, an investigational new drug developed by Henlius (Shanghai China), has passed the Phase I clinical trial, but its huge amount of charge isoforms and complicated charge heterogeneity posed a challenge to charge variant investigation in pharmaceutical development. We employed offline free-flow isoelectric focusing (FF-IEF) fractionation, followed by detailed characterization of enriched ACE2Fc fractions, to unveil the structural origins of charge heterogeneity in ACE2Fc expressed by recombinant CHO cells. We adopted a well-tuned 3-component separation medium for ACE2Fc fractionation, the highest allowable voltage to maximize the FF-IEF separation window and a mild Protein A elution method for preservation of protein structural integrity. Through peptide mapping and other characterizations, we revealed that the intricate profiles of ACE2Fc charge heterogeneity are mainly caused by highly sialylated multi-antenna N-glycosylation. In addition, based on fraction characterization and in silico glycoprotein model analysis, we discovered that the large acidic glycans at N36, N73, and N305 of ACE2Fc were able to decrease the binding activity towards Spike (S) protein of SARS-CoV-2. Our study exemplifies the value of FF-IEF in highly complex fusion protein characterization and revealed a quantitative sialylation-activity relationship in ACE2Fc.

Development of a platform method for rapid detection and characterization of domain-specific post-translational modifications in bispecific antibodies.

Charge heterogeneity is inherent to all therapeutic antibodies and arises from post-translational modifications (PTMs) and/or protein degradation events that may occur during manufacturing. Among therapeutic antibodies, the bispecific antibody (bsAb) containing two unique Fab arms directed against two different targets presents an additional layer of complexity to the charge profile. In the context of a bsAb, a single domain-specific PTM within one of the Fab domains may be sufficient to compromise target binding and could potentially impact the stability, safety, potency, and efficacy of the drug product. Therefore, characterization and routine monitoring of domain-specific modifications is critical to ensure the quality of therapeutic bispecific antibody products. We developed a Digestion-assisted imaged Capillary isoElectric focusing (DiCE) method to detect and quantitate domain-specific charge variants of therapeutic bispecific antibodies (bsAbs). The method involves enzymatic digestion using immunoglobulin G (IgG)-degrading enzyme of S. pyogenes (IdeS) to generate F(ab)2 and Fc fragments, followed by imaged capillary isoelectric focusing (icIEF) under reduced, denaturing conditions to separate the light chains (LCs) from the Fd domains. Our results suggest that DiCE is a highly sensitive method that is capable of quantitating domain-specific PTMs of a bsAb. In one case study, DiCE was used to quantitate unprocessed C-terminal lysine and site-specific glycation of Lys98 in the complementarity-determining region (CDR) of a bsAb that could not be accurately quantitated using conventional, platform-based charge variant analysis, such as intact icIEF. Quantitation of these PTMs by DiCE was comparable to results from peptide mapping, demonstrating that DiCE is a valuable orthogonal method for ensuring product quality. This method may also have potential applications for characterizing fusion proteins, antibody-drug conjugates, and co-formulated antibody cocktails.

Two-Dimensional Gel Electrophoresis in Studies of Flower and Leaf Proteome of Common Buckwheat.

Two-dimensional gel electrophoresis (2-DE) is a proteomic tool used for the separation of protein mixtures according to protein isoelectric point and molecular mass. Although gel-free quantitative and qualitative proteomic study techniques are now available, 2-DE remains a useful analytical tool. The presented protocol was performed to analyze the flower and leaf proteome of common buckwheat using 24 cm immobilized pH gradient strips (pH 4-7) and visualization of proteins on gels via colloidal Coomassie G-250 staining.

CE methods for charge variant analysis of mAbs and complex format biotherapeutics.

Charge heterogeneity analysis of monoclonal antibodies (mAbs) and complex formats, such as bispecifics, is crucial for therapeutic applications. In this study, we developed two capillary electrophoresis (CE)-based methods, capillary zone electrophoresis (CZE) and imaged capillary isoelectric focusing (iCIEF), for analyzing a broad spectrum of mAbs and complex mAb formats. For CZE, we introduced a new buffer system and optimized the background electrolyte (BGE) with an alternative dynamic coating agent and a superior polymeric additive. The pH of the BGE was increased, leading to enhanced resolution of high pI and complex format mAbs. In iCIEF, we identified an ampholyte combination offering a highly linear pH gradient and covering a suitable pH range. We also investigated alternatives to denaturing stabilizers and found that non-detergent sulfobetaine 195 exhibited excellent properties for iCIEF applications. These optimized methods provide a framework for the charge heterogeneity analysis of therapeutic mAbs and complex formats.

Isoelectric focusing followed by affinity immunoblotting to detect monoclonal free light chains in monoclonal gammopathies: Comparison with immunofixation electrophoresis and free light chain ratio.

BACKGROUND: Isoelectric focusing (IEF) is a method with an exquisite resolution, and coupled with affinity immunoblotting (AIB), it can provide superior sensitivity to detect monoclonal free light chains (FLC). METHODS: We tested the hypothesis that IEF/AIB is more sensitive and specific for monoclonal FLC detection in serum and urine samples than conventional methods, that is, electrophoresis (ELP), immunofixation (IF) and serum FLC ratio assessment. Investigation included 107 samples of 68 patients, among which 21 multiple myeloma patients were recently tested for minimal residual disease and 18 patients with AL amyloidosis. RESULTS: Monoclonal FLC were detected by IEF/AIB in 37% of serum samples negative for monoclonal FLC on ELP/IF. As for urine samples, significant advantage of the IEF/AIB over ELP/IF was not demonstrated. Considering both serum and urine results, IEF/AIB definitely revealed monoclonal FLC in 20/83 (24%) of ELP/IF-negative samples. FLC ratio was abnormally high (>1.65) in all 11 patients definitely positive for monoclonal FLC kappa by IEF/AIB but also in 16/47 (34%) IEF/AIB-negative samples. Abnormally low values (<0.26) were found only in 10/28 samples (36%) positive for monoclonal FLC lambda. Appropriate use of renal FLC ratio reference range reduced the number of presumably false positives (6/47, i.e. 13%) but not false negatives (17/28, i.e. 61%). CONCLUSIONS: The IEF/AIB method is more sensitive than IF and might be used in patients with negative IF results before deciding whether to proceed to minimal residual disease testing.

Development and verification of whole column imaging detection-capillary isoelectric focusing method for determination of isoelectric point of pertactin / 中国生物制品学杂志

@#Objective To develop and verify a whole column imaging detection-capillary isoelectric focusing(WCID-CIEF)method for the determination of isoelectric point(pI)of pertactin(PRN).Methods The WCID-CIEF method for the determination of PRN antigen was developed by optimizing the parameters such as the focusing time and final concentration of samples in the WCID-CIEF process,and verified for the specificity,accuracy,repeatability,intermediate precision,durability and inter-batch consistency.Results The optimal focusing time of WCID-CIEF for the determination of PRN antigen pI was 1 min at 1 500 V and 3 min at 3 000 V. The optimal final concentration of PRN antigen was 300 μg/mL. The PRN antigen pI was about 6. 035,and the blank matrix showed no interference peak in the position of each peak of antigen. The method had good specificity,accuracy,repeatability,intermediate precision,durability and consistency among batches.Conclusion The developed WCID-CIEF method is suitable for the pI detection and charge heterogeneity analysis of PRN antigen,which can provide basis for the characterization of PRN antigen and reference for the quality control in the process of development and production of related vaccines.

Mobilization in two-step capillary isoelectric focusing: Concepts assessed by computer simulation.

The mobilization step in a two-step capillary isoelectric focusing protocol is discussed by means of dynamic computer simulation data for systems without and with spacer compounds that establish their zones at the beginning and end of the focusing column. After focusing in an electroosmosis-free environment (first step), mobilization (second step) can be induced electrophoretically, by the application of a hydrodynamic flow, or by a combination of both means. Dynamic simulations provide insight into the complexity of the various modes of electrophoretic mobilization and dispersion associated with hydrodynamic mobilization. The data are discussed together with the relevant literature.

Simple analysis of gel images with IOCBIO Gel.

BACKGROUND: Current solutions for the analysis of Western Blot images lack either transparency and reproducibility or can be tedious to use if one has to ensure the reproducibility of the analysis. RESULTS: Here, we present an open-source gel image analysis program, IOCBIO Gel. It is designed to simplify image analysis and link the analysis results with the metadata describing the measurements. The software runs on all major desktop operating systems. It allows one to use it in either a single-researcher environment with local storage of the data or in a multiple-researcher environment using a central database to facilitate data sharing within the research team and beyond. By recording the original image and all operations performed on it, such as image cropping, subtraction of background, sample lane selection, and integration boundaries, the software ensures the reproducibility of the analysis and simplifies making corrections at any stage of the research. The analysis results are available either through direct access to the database used to store it or through the export of the relevant data. CONCLUSIONS: The software is not only limited to Western Blot image analysis and can be used to analyze images obtained as a part of many other widely used biochemical techniques such as isoelectric focusing. By recording the original data and all the analysis steps, the program improves reproducibility in the analysis and contributes to the implementation of FAIR principles in the related fields.

Analytical Ultracentrifugation-Calibrated Anion-Exchange Chromatography for Sensitive and Intact Determination of Osteopontin in Infant Formula and Dairy Products.

Osteopontin is a crucial protein ingredient that has been applied in fortified dairy products and infant formula. It has great significance to infant gut health and brain development. However, current techniques including enzyme-linked immunosorbent assay and liquid chromatography coupled with mass spectrometry are still facing the bottleneck of low sensitivity and indirect quantification. Moreover, the unavailable certified commercial OPN standard hinders its accurate quantification. Herein, a novel method of anion-exchange chromatography was established to determine OPN concentration in several dairy matrices. The polarity-reversed capillary isoelectric focusing was utilized to measure the exact isoelectric point (pI) to support method development for OPN separation. Analytical ultracentrifugation was used to calibrate the purity of intact OPN to develop an in-house reference standard. The method showed the merits of limits of detection to 0.04 mg/100 g, relative standard deviation of reproducibility <5% for 13 out of 14 tested matrices, and an average recovery rate of 101.3%. This method has shown the potential to be adopted as an international standard method for the quantification of intact OPN in infant formula and dairy products.

Imaged capillary isoelectric focusing tandem high-resolution mass spectrometry using nano electrospray ionization (ESI) for protein heterogeneity characterization.

Recombinant monoclonal antibodies (mAbs) have been spurring the rapid growth of commercial biotherapeutics. During production their charge heterogeneity must be assessed as a critical quality attribute to ensure safety, efficacy, and potency. Although imaged capillary isoelectric focusing (icIEF) is a powerful tool for this process, it could be improved further with tandem high-resolution mass spectrometry (HRMS). In this work, a nano-electrospray ionization (nano-ESI) apparatus was constructed to directly couple icIEF to HRMS. The system was evaluated with the standard NISTmAb, as well as more complex mAb, bi-specific antibody, and fusion protein samples. NISTmAb concentrations as low as 0.25 mg/ml demonstrated excellent sensitivity. There were good repeatabilities at 1 mg/ml with 7.58% and 8.01% RSDs for intention time and MS intensity, respectively, and the HRMS signal showed a strong linearity (R = 0.9983) across different concentrations. Meanwhile, the fingerprinting of the complex samples illustrated the versatility and potential of icIEF-HRMS. icIEF-HRMS developed can provide a comprehensive understanding of the underlying structural modifications that impact protein charge heterogeneity. Compared to the traditional ESI, nano-ESI can significantly improve sensitivity while maintaining a reasonable repeatability and throughput. Furthermore, the interface is much easier to connect, and is compatible with many commercial HRMS instruments.

Separation and fractionation of glutamic acid and histidine via origami isoelectric focusing.

We demonstrated the fractionation of two amino acids, glutamic acid and histidine, separated via isoelectric focusing (IEF) on filter paper folded and stacked in an origami fashion. Channels for electrophoresis were fabricated as circular zones acquired via wax printing onto the filter paper. An ampholyte solution with amphiphilic samples was deposited on all the circle zones, which was followed by folding to form the electrophoresis channels. IEF was achieved by applying an electrical potential between the anodic and cathodic chambers filled with phosphoric acid and sodium hydroxide solutions, respectively. A pH gradient was formed using either a wide-range ampholyte with a pH of 3 to 10 or a narrow-range version with a pH of 5 to 8, which was confirmed by adding pH indicators to each layer. The origami IEF was used to separate the amino acids, glutamic acid and histidine, by mixing with the ampholytes, which were deposited on the layers. The components in each layer were extracted with water and measured by high-performance liquid chromatography using pre-column derivatization with dansyl chloride. The results indicated that the focus for glutamic acid and that for histidine were at different layers, according to their isoelectric points. The origami isoelectric focusing achieved the fractionation of amino acids in less than 3 min using voltage as low as 30 V.

Efficiency of Biological Typing Methods in Maize Hybrid Genetic Purity Estimation.

A high level of genetic purity in crop varieties must be achieved and maintained for agronomic performance, encouraging investment and innovation in plant breeding and ensuring that the improvements in productivity and quality imparted by breeders are delivered to the consumer. Since the success of hybrid seed production is dependent upon the genetic purity of the parental lines, in this study, the experimental F1exp maize hybrid and its parental inbreeds were used as a model system to examine the discriminative power of morphological, biochemical and SSR markers for seed purity assay. The highest number of off-type plants was estimated by morphological markers. According to the comparison of prolamins and albumins banding patterns of parental and derived F1exp seeds, genetic impurities could not be detected. Molecular analysis detected two types of genetic profile irregularity. Beside its use for verifying varieties of maize, report on umc1545 primer pair ability to detect non-specific bands (i.e., off-types), in both the maternal component and F1exp, which is the first report on this issue yet, strongly supports the recommendation of this SSR marker use for more accurate and time-efficient maize hybrids and parental lines genetic pyrity testing.

Imaged capillary isoelectric focusing associated with multivariate analysis: A powerful tool for quality control of therapeutic monoclonal antibodies.

Monoclonal antibodies are increasingly used in cancer therapy. To guarantee the quality of these mAbs from compounding to patient administration, characterization methods are required (e.g. identity). In a clinical setting, these methods must be fast and straightforward. For this reason, we investigated the potential of image capillary isoelectric focusing (icIEF) combined with Principal Component Analysis (PCA) and Partial least squares-discriminant analysis (PLS-DA). icIEF profiles obtained from monoclonals antibodies (mAbs) analysis have been pre-processed and the data submitted to principal component analysis (PCA). This pre-processing method has been designed to avoid the impact of concentration and formulation. Analysis of four commercialized mAbs (Infliximab, Nivolumab, Pertuzumab, and Adalimumab) by icIEF-PCA led to the formation of four clusters corresponding to each mAb. Partial least squares-discriminant analysis (PLS-DA) applied to these data allowed us to build models to predict which monoclonal antibody is analyzed. The validation of this model was obtained from k-fold cross-validation and prediction tests. The selectivity and the specificity of the model performance parameters were assessed by the excellent classification obtained. In conclusion, we established that the combination of icIEF and chemometric approaches is a reliable approach for unambiguously identifying compounded therapeutic monoclonal antibodies (mAbs) before patient administration.

Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity.

Post-translational modifications (PTMs) of adeno-associated virus (AAV) capsid proteins tune and regulate the AAV infective life cycle, which can impact the safety and efficacy of AAV gene therapy products. Many of these PTMs induce changes in protein charge heterogeneity, including deamidation, oxidation, glycation, and glycosylation. To characterize the charge heterogeneity of a protein, imaged capillary isoelectric focusing (icIEF) has become the gold standard method. We have previously reported an icIEF method with native fluorescence detection for denatured AAV capsid protein charge heterogeneity analysis. Although well suited for final products, the method does not have sufficient sensitivity for upstream, low-concentration AAV samples, and lacks the specificity for capsid protein detection in complex samples like cell culture supernatants and cell lysates. In contrast, the combination of icIEF, protein capture, and immunodetection affords significantly higher sensitivity and specificity, addressing the challenges of the icIEF method. By leveraging different primary antibodies, the icIEF immunoassay provides additional selectivity and affords a detailed characterization of individual AAV capsid proteins. In this study, we describe an icIEF immunoassay method for AAV analysis that is 90 times more sensitive than native fluorescence icIEF. This icIEF immunoassay provides AAV stability monitoring, where changes in individual capsid protein charge heterogeneity can be observed in response to heat stress. When applied to different AAV serotypes, this method also provides serotype identity with reproducible quantification of VP protein peak areas and apparent isoelectric point (pI). Overall, the described icIEF immunoassay is a sensitive, reproducible, quantitative, specific, and selective tool that can be used across the AAV biomanufacturing process, especially in upstream process development where complex sample types are often encountered.

Erratum: Comparison of two different canine anti-IgG antibodies for assessment of oligoclonal bands in cerebrospinal fluid and serum of dogs via isoelectric focusing followed by an immunoblot.

[This corrects the article DOI: 10.3389/fvets.2022.873456.].