Alpha1-antitrypsin in serum determined by capillary isoelectric focusing

A capillary isoelectric focusing (CIEF) method using bare fused-silica capillaries filled with polyethylene oxide (PEO) and carrier ampholyte solutions in the pH 3.5-5.0 range has been developed for the identification of alpha1-antitrypsin (alpha1AT) phenotypes in human serum. This novel procedure was routinely applied to the study of serum samples of five controls whose alpha1AT phenotype was previously identified and of twelve subjects whose alpha1AT phenotype was unknown. The results obtained allowed us to confirm or identify the alpha1AT phenotype in all sera tested. This procedure seems particularly suitable for identification of alpha1AT variants associated with diseases of clinical relevance.     

Isoforms analysis of recombinant human erythropoietin by polarity-reversed capillary isoelectric focusing

Recombinant human erythropoietin (rhuEPO) has been extensively used as a pharmaceutical product for treating anemia in the clinic. Glycosylation of rhuEPO was crucial for affecting biological activity, immunogenicity, and pharmacokinetics. Because of the heterogeneity of glycan, the structure of rhuEPO was complex with several isoforms. Characterization of isoforms was important for quality control of rhuEPO. Here, an improved cIEF method has been established and validated. A polarity-reversed focusing step was used by reversing both the polarity of the voltage and the catholyte and anolyte vials. A weak base (100 mM ammonium hydroxide solution) was used as a chemical mobilizer to make the acidic bands mobilize stably to the detection window. Compared with CZE method in European Pharmacopoeia, the numbers of isoforms and their peak area percentage were highly consistent. Better reproducibility and higher resolution have been obtained by the improved cIEF method. Moreover, in improved cIEF method, the isoelectric points (pI) of each isoform can be calculated and used for identification. It was also the first time that the cIEF method was fully validated for rhuEPO analysis according to the International Conference on Harmonization (ICH) guidelines.     

Validation of a capillary isoelectric focusing method for the recombinant monoclonal antibody C2B8

A capillary isoelectric focusing (cIEF) method has been developed for the purpose of determining the identity and charge distribution of mouse/human chimeric antibody to human CD20 antigen (C2B8). The assay was validated in accordance with ICH guidelines in order to demonstrate that it is suitable for its intended purpose and so that it may be performed as a lot release test for bulk and final product. As a result of the validation process the assay was found to be linear over the concentration range of 2–356 μg ml⁻¹ with recovery of ¹²⁵I-labeled C2B8 at the target sample concentration of 125 μg ml⁻¹ equal to 99%. The repeatability and intermediate precision relative standard deviations of the four major peaks for migration time, peak area, and peak area percent ranged from 0.9–4.4%. The specificity of the assay was demonstrated by baseline resolution of the C2B8 main peak from product excipients, and other Genentech monoclonal antibodies. The results of this validation demonstrate that the cIEF assay for the determination of identity and charge distribution of C2B8 is accurate, precise, linear, and highly specific. The assay is rapid and suitably rugged.     

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.     

Changes of alpha1-acid glycoprotein microheterogeneity in acute inflammation stages analyzed by isoelectric focusing using serum obtained postoperatively

The relationship between variations of alpha1-acid glycoprotein (orosomucoid, AGP) microheterogeneity detected from isoelectric focusing (IEF) patterns and clinical stage of acute inflammation based on serum C-reactive protein (CRP) levels and interleukin-6 (IL-6) levels was investigated. Serum samples were obtained from healthy subjects, and from patients with esophageal or stomach carcinoma before and after operation. Samples without neuraminidase treatment were used for AGP microheterogeneity analysis, and samples with neuraminidase treatment for AGP heterogeneity analysis. In AGP microheterogeneity, nine bands were detected in the range of pI 3.18-3.57 in sera obtained from healthy subjects. In patients, AGP microheterogeneity changed the first day after operation; the percentage of bands surrounding pI 3.5 increased, and the highest value appeared in sera taken the first or second day after operation and then decreased quickly. These bands showed reactivity for concanavalin A (Con A). The increase in Con A-reactive AGP occurred later than the increase in IL-6, and occurred earlier than the increase in CRP. On the seventh day after operation, the percentage of bands around pI 3.2 increased. These bands showed the reactivity for Datura stramonium agglutinin. On the other hand, in samples with neuraminidase treatment, little change of AGP heterogeneity was observed in most samples, which did not reflect the stage of inflammation. These findings suggested that AGP microheterogeneity detection was a useful marker for the clinical stage of inflammation.     

Fractionation of the human recombinant tissue plasminogen activator (rtPA) glycoforms by high-performance capillary zone electrophoresis and capillary isoelectric focusing.

This paper reports the fractionation of recombinant human tissue plasminogen activator (rtPA) glycoforms, a complex mixture to demonstrate the high resolving power of capillary zone electrophoresis (CZE) and capillary isoelectric focusing (cIEF). rtPA is a glycoprotein with a complex carbohydrate structure. The electropherograms and IEF patterns have been discussed in light of the known carbohydrate structures of rtPA. rtPA was treated with neuraminidase which removes the sialic acids from the carbohydrate chains. The desialylated rtPA was analyzed by both CZE and IEF and the results were compared to those of untreated rtPA. The usefulness of CZE and cIEF in the characterization of glycoproteins proteins is also discussed.     

Human alpha-1-antitrypsin subtyping by hybrid isoelectric focusing in miniaturized polyacrylamide gel

The polymorphism of alpha-1-antitrypsin (PI) has been studied by hybrid isoelectric focusing in miniaturized immobilized pH gradient gels, with an interelectrode distance of 55 mm, in two narrow ranges of pH 4.35-4.75 and 4.35-4.55), following rehydration with pH 4.2-4.9 carrier ampholytes. The use of the separator N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) in combination with carrier ampholytes for gel rehydration has been shown to enhance PI band sharpness. The influence of different additives (sucrose, sorbitol and glycerol) on the PI band pattern has also been evaluated. Glycerol has been shown to be responsible for the change in the relative mobility of the M3 band. The analysis of the minor M-7 isoprotein zone by hybrid isoelectric focusing followed by silver staining has permitted a more reliable classification of PIM subtypes. A population study carried out with 164 unrelated individuals living in Spain is also presented.     

Dual-detection approach for a charge variant analysis of monoclonal antibody combination products using imaged capillary isoelectric focusing

The clinical benefits of treatments with a combination of two or more therapeutic monoclonal antibodies (mAbs) have emerged in recent years. Imaged capillary isoelectric focusing is a frequently used technology in the biopharmaceutical industry for charge variant analysis of protein therapeutics. However, with the wide concentration ranges of combination products, one component may fall within the linear detection range, whereas the other does not. Here, we report a novel methodology to explore charge variants of mAb mixtures using multiple detection techniques simultaneously. We use ultraviolet absorbance to monitor the charge variants of the high-concentration component and native fluorescence (FL) to monitor the variants of the low-concentration one. Charge variants of mixtures that span 40-fold in ratio differences can be accurately quantified with this approach. In contrast to the conventional methods, it is not necessary to prepare and analyze two samples at different concentrations and combine the results for combination product testing. Additionally, the use of FL detection enables the charge variant analysis of highly potent/low abundant mAbs in a mixture. This methodology is more quality-control friendly and efficient for the charge variant analysis of combination products with wide ratios.     

Element of a validation method for MU-B3 monoclonal antibody using an imaging capillary isoelectric focusing system

This paper presents an imaging capillary isoelectric focusing (CIEF) assay for the determination of the identity, stability, and isoform distribution of a murine monoclonal antibody (MU-B3). The experiments were conducted using a Convergent Bioscience iCE280 instrument. The optimum carrier ampholyte composition that gave the best peak separation was found to be 25% Pharmalyte pH 3-10 and 75% Pharmalyte pH 5-8. The antibody gave a highly reproducible CIEF profile with three major peaks having average isoelectric point (pI) values of 6.83, 6.99, and 7.11. Intraday and interday reproducibility of pI values was found to be within RSD of 0.5%. The CIEF profile was also the same, with an alternate column cartridge and alternate batches of methyl cellulose. A plot of peak areas versus MU-B3 concentration was linear (R2 = 0.995) up to a concentration of 0.5 mg/mL in the sample solution. Peak area measurements were reproducible to within 7% RSD. The CIEF profiles of two other antibodies were distinctly different from the profile of MU-B3, showing that the assay is specific. After a sample of MU-B3 was subjected to heat stress by exposure to heat at 55 degrees C for 4 h, its CIEF profile was altered with extra peaks appearing at lower pI values, indicating that the assay could be used to monitor stability. The result of the heat stress experiment was also confirmed with a parallel slab-gel IEF analysis of the antibody sample before and after application of the heat stress. The results of this work suggest that imaging CIEF can be used for product testing under a quality control environment. The assay can be used for pI profiling of proteins and for monitoring structural changes (deamidation, glycosylation, etc.) during the manufacturing process and upon storage.     

Determination of the isoelectric point of proteins by capillary isoelectric focusing

Different ways of determining isoelectric points (pI) of proteins in capillary isoelectric focusing are reviewed here. Due to the impossibility of direct pH measurements in the liquid phase, such assessments have to rely on the use of pI markers. Different types of pI markers have been described: dyes, fluorescently labelled peptides, sets of proteins of known pI values. It appears that, perhaps, the best system is a set of 16 synthetic peptides, trimers to hexamers, made to contain each a Trp residue for easy detection at 280 nm. By a careful blend of acidic (Asp, Glu), mildly basic, with pK around neutrality (His), and basic (Lys, Arg) amino acids, it is possible to obtain a series of pI markers with pI values quite evenly distributed along the pH scale, possessing good buffering capacity and conductivity around their pI values and thus focusing as sharp peaks. Another approach to pI determination is the monitoring of the current during mobilization: this allows, with the aid of known pI markers, to calibrate the system with a pI/current graph. Pitfalls and common errors in pI determinations are reviewed here and guidelines given for minimizing such errors in pI estimation.     

Enantioseparation of derivatized amino acids by capillary isoelectric focusing using cyclodextrin complexation

Enantioseparation of dansylated as well as 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC)-derivatized amino acids by means of capillary isoelectric focusing using various cyclodextrin derivatives is demonstrated. Separation is based on the enantioselective shift of the isoelectric points upon complexation with the chiral selectors. The zwitterionic, diastereomeric analyte-cyclodextrin complexes exhibited differences in the pI values up to more than 0.25 pI units. Enantioresolution was achieved for a number of derivatized amino acids and various selectors added to the carrier ampholyte solution. The hydroxypropyl-beta-cyclodextrin proved to be the best selector for this purpose. Enantioseparation as dependent on the selector concentration was evaluated in a range between 5 and 30 mM. Separation could be attained down to selector concentrations corresponding to a degree of complexation as low as 30%. The peaks appear according to the degree of complexation between the positions adopted without and with full complexation. The kinetics of complex formation and dissociation was fast enough in most instances to produce single peaks, even with complexation degrees near 0.5 and significant pI shifts. Peak widths were slightly enlarged in these instances. The method offers excellent perspectives for preparative applications.     

Characterization of bovine serum albumin-tryptophan interaction by capillary isoelectric focusing with whole column imaging detection

Capillary isoelectric focusing (cIEF) with whole column imaging detection (WCID) was explored for the characterization of bovine serum albumin (BSA)-tryptophan interaction, to further understand protein-drug interactions. The BSA-tryptophan interaction was dynamically monitored by cIEF-WCID, to provide the cIEF profiles of the BSA-tryptophan interaction system at different focusing times. Our study demonstrated that the cIEF behavior of BSA can serve as a probe into the study of BSA-tryptophan interaction, through monitoring the change in its cIEF profile when the interaction occurred. The study illustrated that the BSA peak split due to the BSA-tryptophan interaction, and the peak of BSA-tryptophan complex was clearly identified in the cIEF electropherograms. By comparing the cIEF profiles of BSA/L-tryptophan and BSA/D-tryptophan, respectively, our study demonstrated that BSA interacted with the enantiomers of tryptophan with a chiral recognition. L-Tryptophan demonstrated a very strong interaction with BSA, while D-tryptophan exhibited a much weaker interaction with BSA. The effects of the BSA concentration, the tryptophan concentration, the focusing time and the incubation time on the BSA-tryptophan interaction were investigated. This study offers a novel approach for the study of protein-drug interactions.     

Application of immobilized pH gradient isoelectric focusing to forensic hemogenetics: a survey on a three year experience with the transferrin (TF) and alpha 1-antitrypsin (PI) systems

The subtypes of transferrin (TF) and alpha 1-antitrypsin (PI), first discovered using isoelectric focusing, are now mostly determined in immobilized pH gradient gels. We report on our experience in the parentage expertise with both polymorphisms over a period of three years. The complexity of the technology was compensated by the fact that most subtypes of TF and PI could be more reliably recognized. The PI alleles PI*M1, M2, M3, S, F, T, and Z and TF alleles TF*C1, C2 and C3, and in addition four further rare TF alleles were observed. The allele frequencies from non-related individuals did not deviate from the Hardy-Weinberg equilibria and corresponded well to known frequencies from West Germany and other Caucasoid populations. With the TF system 36 accused men, and with the PI system 54 were excluded from paternity from a total of 344 (TF) respectively 347 (PI) cases. From the data presented here isoelectric focusing in immobilized pH gradient gels appears to be a major improvement over carrier ampholyte generated pH gradients in the distinction of TF and PI phenotypes.     

Analysis of acrylamido-buffers for isoelectric focusing by capillary zone electrophoresis

Immobilized pH gradients use a series of weak acrylamido acids and bases (Immobiline) to create a pH gradient along the separation axis. These buffers can be degraded in water by two mechanisms: (i) hydrolysis of the amido bond, with generation of free acrylic acid and either an amino acid or a diamine; (ii) autopolymerization to oligomers and/or n-mers. In order to check for these degradation products, different capillary zone electrophoresis systems for analysis of all Immobilines have been devised. The acidic compounds are resolved in 100 mM acetate, pH 4.0, whereas the alkaline Immobilines are separated in 50 mM phosphate buffer, pH 7.7 (or pH 7.2 for the weaker species). Polymers of alkaline Immobilines are resolved in 50 mM phosphate buffer, pH 2.5, in 1% Ficoll-400. All Immobilines are detected underivatized, by their adsorption at 214 or 254 nm. A calibration curve has been constructed for quantification of acrylic acid contamination. As little as 1 mol% of acrylic acid contamination in Immobiline solutions can be detected, with a sensitivity limit below 0.2 mM (at the injection port).     

Analysis of genetic variants of transferrin in human serum after desialylation by capillary zone electrophoresis and capillary isoelectric focusing

Capillary electrophoresis analysis of transferrin in human serum is used to assess genetic variants after desialylation with neuraminidase and iron saturation to reduce the complexity of the transferrin pattern and thus facilitate the recognition of transferrin polymorphisms. Asialo‐transferrin forms are analyzed by capillary zone electrophoresis using assay conditions as for the monitoring of carbohydrate‐deficient transferrin or by capillary isoelectric focusing in a pH 5–8 gradient which requires immunoextraction of transferrin prior to analysis. With the carrier ampholytes used, peaks for iron saturated and iron depleted transferrin are monitored which indicates complexation of iron ions by carrier ampholytes. For BC, CD, and BD genetic variants, the expected peaks for B, C, and D forms of transferrin were detected with both methods. Monitoring of CC patterns revealed three cases, namely those producing double peaks in both methods, a double peak in capillary isoelectric focusing only and a double peak in capillary zone electrophoresis only. For all samples analyzed, data obtained by capillary isoelectric focusing could be confirmed with gel isoelectric focusing. The two capillary electrophoresis methods are shown to represent effective tools to assess unusual transferrin patterns, including genetic variants with dissimilar abundances of the two forms.     

Validatibility of a capillary isoelectric focusing method for impurity quantitation

A strategy is presented for examining the validatability of a capillary isoelectric focusing (cIEF) method, intended for quantitation of product-related impurities in a protein drug substance, according to guidelines published by the International Conference on Harmonization (ICH). The results of this study demonstrate the suitability of cIEF as an analytical method for the quantitation of two product-related impurities in a protein drug substance: a monodeamidated degradation product and an aggregated form of the parent molecule. A range of impurity levels was generated by spiking the isolated impurity species, into a representative production lot of the drug substance. Six impurity spike levels (0.5-12% impurity for deamidated species and 0.5-8% impurity for aggregated species) were analyzed in triplicate. Measurement of impurity peak area percent in the spiked samples provided the data for computing specificity, accuracy, precision, linearity and limit of quantitation (LOQ) for the impurities. Accuracy, defined as the agreement of peak area percent for impurity species with the theoretical impurity percentage from the spike ratio, was 85-96% for the deamidated species and 73-97% for the aggregated species. A linear relationship was found between the measured area percent and the theoretical percent impurity for both impurity species (coefficient of determination, r2=0.9994 for deamidated species and =0.9827 for aggregated species). Precision (repeatability) studies demonstrated a low relative standard deviation (RSD) value (<6%) at all spike levels for both impurity species. Intermediate precision and reproducibility were evaluated by simulating many of the multivariable testing conditions expected during the life cycle of an analytical method, such as multiple equipment and laboratories. Repeated analyses of the drug substance under these varied conditions, yielded RSD values of <20%, for both impurity species. The LOQ, defined as the lowest impurity level where both accuracy and precision were achieved, was assigned at the 0.5% impurity level for both impurity species. This work illustrates a successful strategy in applying the ICH validation guidelines for impurity analytical methods to a cIEF method. Moreover, the data demonstrate the ability of cIEF to be used reliably as an analytical method for impurity quantitation.     

Reliable phenotyping of alpha-1-antitrypsin by hybrid isoelectric focusing in an ultranarrow immobilized pH gradient.

Genetically determined phenotypes of the highly polymorphic human alpha 1-antitrypsin were examined by hybrid isoelectric focusing in a narrow immobilized pH gradient. The chosen pH range from 4.45 to 4.75 was useful for identification and classification of the common PI M subtypes and a number of PI variants in the microheterogeneous regions of m6, m7, and m8. A high degree of resolution and an improved sharpness of PI bands was achieved with this excellent technique. It allowed the distinction of a new PI M variant, which has been designated M8, or Mingolstadt, according to the PI nomenclature. The pI difference of this mutant to the slightly cathodically located subtype M3 is approximately 0.001 pH unit. In addition, some common as well as rare phenotypes are presented.     

Peak identification in capillary isoelectric focusing using the concept of relative peak position as determined by two isoelectric point markers

In CIEF analysis, sample peaks can be identified by their relative peak positions (RPP) that are determined using only two internal pI markers. The two internal pI marker peaks should bracket, as close as possible, the sample peaks. The RPP values of the sample peaks are then calculated using the pI values, peak positions of the two pI markers, and peak position of the sample. Use of this method can effectively compensate for pH gradient distortions that often occur as a result of salts. Also, as shown by the results of this paper, regardless of the linearity of the pH gradient established by the given carrier ampholytes, sample peaks can be identified within an SD of 0.1 pH unit in RPP (<2% RSD) as long as the sample is run using the same carrier ampholytes and maintaining salt concentrations in the range of 0-15 mM.     

Charge profiling and stability testing of biosimilar by capillary isoelectric focusing

CIEF was developed for the rapid analysis of charge heterogeneity of trastuzumab biosimilar using commercially available fluorocarbon‐coated capillary. The CIEF master mix was composed of 0.30% w/v methyl cellulose, 2.3 M urea, 56.8 mM l ‐arginine, 1.52 mM iminodiacetic acid, 4.5% v/v carrier ampholytes (broad‐range pI 3–10 and narrow‐range pI 8–10.5 with ratio of 3:1), and 0.45% v/v 10.0, 9.5, 7.0, 5.5, 4.1 pI markers. To get a robust method to analyze charge heterogeneity, some separation parameters, including focusing time and separation temperature, were investigated and optimized. The optimized method gave good precision in estimated pI values of charge variants with RSDs of not more than 0.16% intraday analysis (n = 6) and < 0.18% interday analysis (n = 9). In addition, the applications of this method including purity, stability, lot consistency, peptide N‐glycosidase F digest, and C‐terminal lysine variants characterization were also investigated.     

Rapid and high-resolution glycoform profiling of recombinant human erythropoietin by capillary isoelectric focusing with whole column imaging detection

Human erythropoietin (hEPO) is a glycoprotein hormone produced primarily by the kidney, which stimulates red blood cell production. Recombinant human erythropoietin (rhEPO), generally produced in Chinese hamster ovary (CHO) cells, can be used as not only a therapeutic protein but also a doping agent in sports. Profiling of EPO glycoforms is a critical means for quality control in pharmaceutical industrial and anti-doping analysis of misuse in sports. However, the existing methods for the analysis of EPO are associated with either time consuming or poor resolution. In this work, a rapid and high-resolution glycoform profiling method was presented based on capillary isoelectric focusing (cIEF) with whole column imaging detection (WCID). Experimental conditions that influence the separation were investigated. Under optimized conditions, rhEPO from three different sources were resolved into distinct populations within 5 min with excellent reproducibility. As compared with existing methods, the presented method exhibited the advantages of speed and high resolution. If combined with an effective sample enrichment step and a much more sensitive WCID version, the method can be a potential alternative for the detection of rhEPO misuse in sports.