Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.     

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.     

Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography

A collaborative study on the robustness and portability of a capillary electrophoresis‐mass spectrometry method for peptide mapping was performed by an international team, consisting of 13 independent laboratories from academia and industry. All participants used the same batch of samples, reagents and coated capillaries to run their assays, whereas they utilized the capillary electrophoresis‐mass spectrometry equipment available in their laboratories. The equipment used varied in model, type and instrument manufacturer. Furthermore, different types of sheath‐flow capillary electrophoresis–mass spectrometry interfaces were used. Migration time, peak height and peak area of ten representative target peptides of trypsin‐digested bovine serum albumin were determined by every laboratory on two consecutive days. The data were critically evaluated to identify outliers and final values for means, repeatability (precision within a laboratory) and reproducibility (precision between laboratories) were established. For relative migration time the repeatability was between 0.05 and 0.18% RSD and the reproducibility between 0.14 and 1.3% RSD. For relative peak area repeatability and reproducibility values obtained were 3–12 and 9–29% RSD, respectively. These results demonstrate that capillary electrophoresis‐mass spectrometry is robust enough to allow a method transfer across multiple laboratories and should promote a more widespread use of peptide mapping and other capillary electrophoresis‐mass spectrometry applications in biopharmaceutical analysis and related fields.     

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.     

Capillary isoelectric focusing--reproducibility and protein adsorption

Capillary isoelectric focusing (CIEF) is an important tool for the quality assurance of biotechnologically maintained drugs and for proteome analysis. The critical performance parameters of this technique are the precisions of isoelectric point (pI) values and peak areas. Compared to capillary zone electrophoresis (CZE), where precise results can be obtained (e.g., 0.5% relative standard deviation (RSD) for peak areas, n = 60), only few data are available for CIEF experiments. So far, reproducible data of pI values (RSD = 0.5%) have been acquired, but peak areas show inferior results (about 3-15% RSD). Nonstable capillary coatings and protein adsorption have been discussed as possible reasons. Recent work of Righetti et al. [25, 27] has proven that the use of coated capillaries can reduce the adsorption of proteins by 50% but cannot prevent it. In our CIEF experiments irregular and poorly reproducible peak patterns have been observed. In a long-time experiment of 106 repeated runs, an overall RSD of 10% was obtained for peak areas, RSD of 2% only in series of about 10 consecutive replicates. Especially at higher concentrations the reproducibility deteriorates. This seems to be the result of a self-amplifying process, induced by adsorbed protein molecules, leading to further agglomerations. CZE control experiments in linear polyacrylamide (LPA)-coated capillaries proved a strong pH dependency of these effects within a small range. Compared to bare fused-silica surfaces, adsorption effects are reduced but not inhibited. An enhancement of reproducibility in CIEF experiments can be achieved only by controlling the interactions of proteins and capillary walls.     

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.     

A Series of Collaborations Between Various Pharmaceutical Companies and Regulatory Authorities Concerning the Analysis of Biomolecules Using Capillary Electrophoresis

An international project team (including members from US, Canada and UK) has been formed from a number of interested biopharmaceutical companies and regulatory authorities to conduct a cross-organisation collaboration exercise. The results from this exercise demonstrate the robustness of CE-SDS across eight different organisations that used instruments of the same equipment model, the same reagents, and the same methodology. Data generated from the analysis of a series of molecular weight markers showed very good precision with regards to relative migration time (RMT) both within and between organisations. The apparent molecular weight of bovine serum albumin (BSA) was measured with good precision to within approximately 2% RSD across the participants. A representative IgG sample showed similar results with regards to relative migration time of its 3 main components, IgG Light Chain, IgG Non-glycosylated Heavy Chain, and IgG Heavy Chain. Fractional peak area for each peak also showed good agreement, with less than 9% RSD for all peaks. This exercise will facilitate both increased regulatory and industrial opinion of CE for biopharmaceutical analysis.CE in the Biotechnology & Pharmaceutical Industries: 7th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, Montreal, Canada, August 12–16, 2005     

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.     

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.     

CIEF with hydrodynamic and chemical mobilization for the separation of forms of alpha-1-acid glycoprotein

Alpha-1-acid glycoprotein (AGP) is a protein that exists in different forms, which is due to variations in the amino acid sequence and/or in the glycosidic part of the protein. These differences confer to these forms, among other characteristics, diverse pIs. Changes in these forms of AGP have been correlated to modifications of the pathophysiological conditions of the individuals. One of the analytical techniques employed for their study has been IEF performed in slab gels. CIEF method with hydrodynamic and chemical mobilization, involving an isotachophoretic process, is developed in this work to separate up to 12 bands of forms of standard AGP, which is proposed as a more reproducible, quantitative, less sample-consuming, and more automated one than conventional IEF. The challenge of this work has been the development of a CIEF method for the separation of bands of a very acidic protein (pI range: 1.8-3.8) in a capillary. Intraday RSD values < or = 1.7% have been achieved for the relative migration time of the AGP bands to that of an internal standard. For intraday area precision, RSD (%) in the range of 2.70-22.71% for AGP zones accounting for more than 10% of total area of AGP sample has been obtained. As a proof of the potential of the methodology proposed, an AGP sample purified from a pool of sera of patients suffering from ovary cancer is analyzed by CIEF.     

Estimation of isoelectric points of human plasma proteins employing capillary isoelectric focusing and peptide isoelectric point markers

Synthetic UV-detectable peptide pI markers were used to estimate isoelectric point (pI) values of proteins separated by capillary isoelectric focusing (CIEF) followed by cathodic mobilization in the absence of denaturing agents. The pI calculation and quantitative analysis of purified proteins showed the feasibility of these peptides as pI markers and internal standards in CIEF separation of proteins. Estimation of pI values of major proteins in human plasma was performed using the peptide pI markers, and the values were compared with those previously obtained by gel isoelectric focusing (IEF). Sera of immunoglobulin G (IgG) myeloma patients, which showed characteristic peaks of myeloma IgG in their CIEF patterns, were also subjected to the analysis and the pI values of the myeloma proteins have been estimated.     

Separation of hemoglobin variants with similar charge by capillary isoelectric focusing: value of isoelectric point for identification of common and uncommon hemoglobin variants

Clinical assays for the primary evaluation of congenital hemoglobin (Hb) disorders must detect and identify a variety of Hb variants. We analyzed hemolysates containing Hb variants with similar charge to evaluate the diagnostic sensitivity and specificity of automated capillary isoelectric focusing (CIEF). Peak separation was observed for each variant in samples containing Hb S, D, and G. The calculated isoelectric points (pI) of these variants were significantly different such that each could be identified in a single run with pI as the sole criterion of identification. The pI of Hb C was significantly different from that of Hb E, C-Harlem, and O-Arab. Hb E, C-Harlem, and O-Arab had similar pI and were not readily differentiated. Hb Koln, M-Saskatoon, Aida, and S/Aida hybrid were readily separated from common Hb variants and detected by CIEF. We conclude that CIEF exhibits both diagnostic sensitivity and specificity, and that pI is an objective and specific criterion of Hb variant identification.     

Interlaboratory method validation of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) methodology for analysis of mAbs

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is an analytical method to assess the purity of proteins, commonly applied to monoclonal antibodies (mAbs) in the biopharmaceutical industry. To address the need to standardize the CE-SDS method in the pharmaceutical industry and to enhance the confidence in method transfer between laboratories operating different commercial capillary electrophoresis (CE) instrument platforms, an interlaboratory CE-SDS method validation was organized involving 13 laboratories in 13 companies on four different types of commercial capillary electrophoresis instruments. In the validation, a commercial mAb therapeutic was used as the sample. The validation process followed the analytical guidelines set by the ICH guidelines (International Conference for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use). The method's precision, accuracy, linearity and range, and limit of quantitation (LOQ) were validated in the study. Variations of all the parameters validated in the study passed the pre-set criteria defined at the beginning of the study. The definition was based on previously published works and the intended application purpose of the CE-SDS method for mAbs. The study proved that the CE-SDS method fits its intended application purpose as a size impurity assay and size heterogeneity characterization assay for mAb therapeutic products. This study is the first time a CE-SDS method is validated by multiple laboratories using different commercial CE instrument platforms and on a commercial mAb therapeutic. Its results will enhance the confidence of the biopharmaceutical industry to develop CE-SDS methods and transfer CE-SDS methods between different laboratories.     

Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection

Sixteen peptides (trimers to hexamers) were designed for use as a set of pI markers for capillary isoelectric focusing (CIEF). Each peptide contains one tryptophan residue for detection by UV absorption and other amino acid residues having ionic side chains, which are responsible for focusing to its pI. The pIs of these peptides were determined by slab-gel IEF using commercial carrier ampholytes. The focused peptides in the gel were detected by absorption measurement at 280 nm using a scanning densitometer and the pH gradient was determined by measuring the pH of the gel using an oxidized metal membrane electrode. The pI values of the peptides ranged from 3.38 to 10.17. The obtained values agreed well with the predicted ones, which were calculated based on amino acid compositions, with root mean square differences of 0.15 pH unit. The peptides were detected at 280 nm as very sharp peaks when separated by CIEF. The pI values of some standard proteins were redetermined by CIEF by using this set of peptide pI markers and the values agreed closely with those reported previously. The sharp focusing, stability, high purity and high solubility of these synthetic pI markers should facilitate the profiling of a pH gradient in a capillary and the determination of the pI values of proteins.     

A Series of Collaborations between Various Pharmaceutical Companies and Regulatory Authorities Concerning the Analysis of Biomolecules Using Capillary Electrophoresis: Additional Instruments/Buffer

An international project team (including members from US, Canada and UK) was formed from a number of interested biopharmaceutical companies and regulatory authorities to conduct a cross-organisation collaboration exercise. The results of the first comparison with eight different organisations that used instruments of the same equipment model, the same reagents, and the same methodology has been reported previously [1]. This report represents the addition of other instruments using a different run buffer. The relative migration times were different, as expected, prohibiting a direct comparison between companies. The within-organisation variability was low for both relative migration time (<0.34% RSD% for all companies save one) and the peak area (<5% RSD% for all companies save one) when measuring the purity of a representative IgG sample. The apparent molecular weight of bovine serum albumin was measured with good precision (less than 10% RSD% across all companies) to the theoretical value when all data is utilized (67.5 kDa compared to 66.4 kDa). For a representative IgG sample, the three main components, IgG Light Chain, IgG Non-glycosylated Heavy Chain, and IgG Heavy Chain, could not be separated, specifically the IgG Non-glycosylated Heavy Chain and IgG Heavy Chain. When the IgG Non-glycosylated Heavy Chain and IgG Heavy Chain were combined for all organisations, the fractional peak area for the IgG Light Chain and IgG Non-glycosylated Heavy Chain + IgG Heavy Chain peak also showed excellent agreement, with less than 7.5 and 3.5% RSD%, respectively. The value of this exercise is in demonstrating the reliability of CE for the determination of apparent size of biopharmaceutical proteins. This underpins the appropriate use of such CE data in support of regulatory submissions.     

Interference‐free mass spectrometric detection of capillary isoelectric focused proteins,including charge variants of a model monoclonal antibody

CIEF represents an elegant technique especially for the separation of structural similar analytes, whereas MS is a state‐of‐the‐art instrumentation for the identification and characterization of biomolecules. The combination of both techniques can be realized by hyphenating CIEF with CZE‐ESI‐MS applying a mechanical valve. During the CZE step, the remaining ESI‐interfering components of the CIEF electrolyte are separated from the analytes prior to MS detection. In this work, a multiple heart‐cut approach is presented expanding our previous single heart‐cut concept resulting in a dramatical reduction of analysis time. Moreover, different sample transfer loop volumes are systematically compared and discussed in regard to peak width and transfer efficiency. With this major enhancement, model proteins (1.63–9.75 mg/L), covering a wide pI range (5–10), and charge variants from a deglycosylated model antibody were analyzed on intact level. The promising CIEF‐CZE‐MS setup is expected to be applicable in different bioanalytical fields, e.g. for the fast and information rich characterization of therapeutic antibodies.     

Characterization of therapeutic mAb charge heterogeneity by iCIEF coupled to mass spectrometry (iCIEF–MS)

Imaged capillary isoelectric focusing (iCIEF) has emerged as an important technique for therapeutic monoclonal antibody (mAb) charge heterogeneity analysis in the biopharmaceutical context, providing imaged detection and quantitation by UV without a mobilization step. Besides quantitation, the characterization of separated charge variants ideally directly by online electrospray ionization–mass spectrometry (ESI–MS) is crucial to ensure product quality, safety, and efficacy. Straightforward direct iCIEF–MS coupling combining high separation efficiency and quantitative results of iCIEF with the characterization power of MS enables deep characterization of mAb charge variants. A short technical setup and optimized methodical parameters (30 nl/min mobilization rate, 2%–4% ampholyte concentration, 0.5–2 mg/ml sample concentration) allow successful mAb charge variant peak assignment from iCIEF to MS. Despite a loss of separation resolution during the transfer, separated intact mAb charge variants, including deamidation as well as major and minor glycoforms even from low abundant charge variants, could be characterized by online ESI–MS with high precision. The presented setup provides a large potential for mAb charge heterogeneity characterization in biopharmaceutical applications.     

Isoelectric point determination by imaged CIEF of commercially available SARS-CoV-2 proteins and the hACE2 receptor

In order to contribute to the scientific research on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we have investigated the isoelectric points (pI) of several related proteins, which are commercially available: the receptor-binding domain (RBD) with His- and Fc-tag, the S1 subunit with His-tag, the S1/S2 subunits with His-tag and the human angiotensin-converting enzyme 2 (hACE2) with His-tag. First, the theoretical pI values, based on the amino acid (AA) sequences of the proteins, were calculated using the ProtParam tool from the Bioinformatics Resource Portal ExPASy. The proteins were then measured with the Maurice imaged CIEF system (native fluorescence detection), testing various measurement conditions, such as different ampholytes or ampholyte mixtures. Due to isoforms, we get sections with several peaks and not just one peak for each protein. The determined pI range for the RBD/Fc is 8.24–9.32 (theoretical pI: 8.55), for the RBD/His it is 7.36–9.88 (8.91) and for the S1/His it is 7.30–8.37 (7.80). The pI range of the S1/S2/His is 4.41–5.87 (no theoretical pI, AA sequence unknown) and for hACE2/His, the determined global range is 5.19–6.11 (5.60) for all experimental conditions chosen. All theoretically derived values were found within these ranges, usually close to the center. Therefore, we consider theoretical values as useful to make predictions about the isoelectric points of SARS-CoV-2 proteins. The experimental conditions had only a minor influence on the pI ranges obtained and mainly influenced the peak shapes.     

Characterization of human papillomavirus by capillary isoelectric focusing with whole‐column imaging detection

CIEF with whole‐column imaging detection (WCID) can be a useful tool for the characterization and identification of human papillomavirus (HPV). This article is the initial report of the determination of the pI of HPV by CIEF‐WCID method. In this study, components of the assay selected for optimization were ampholytes, additives, methylcellulose concentration, HPV concentration, salt concentration, and focusing time. Then the optimization CIEF‐WCID method was validated for HPV 16L1 and HPV 18L1. As a result, a precise method to analyze the pI values of HPVs was achieved with RSD < 1.0%. The HPV peak pattern was reproducible. CIEF‐WCID had great potential for HPV quality control, as WCID eliminated the mobilization step required by the conventional single‐point detection. In the example, the five HPVs displayed pI values of 8.43 ± 0.06 (n = 10; HPV 6L1), 8.70 ± 0.04 (n = 10; HPV 11L1), 7.94 ± 0.05 (n = 18; HPV 16L1), 7.57 ± 0.04 (n = 18; HPV 18L1), and 8.45 ± 0.05 (n = 10; HPV 58L1). This CIEF‐WCID platform could be a powerful analytical tool for characterization, process development support, release testing, and stability study in pharmaceutical industry.     

Rapid determination of fumonisins in corn-based products by liquid chromatography/tandem mass spectrometry

A simple, fast, and robust method was developed for the determination of fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3) in corn-based human food and animal feed (cornmeal). The method involves a single extraction step followed by centrifugation and filtration before analysis by ultra-performance liquid chromatographylelectrospray ionization (UPLC/ESI)-MS/MS. The LC/MS/MS method developed here represents the fastest and simplest procedure (<30 min) among both conventional HPLC methods and other LC/MS methods using SPE cleanup. The potential for high throughput analysis makes the method particularly beneficial for regulatory agencies and analytical laboratories with a high sample volume. A single-laboratory validation was conducted by testing three different spiking levels (200, 500, and 1000 ng/g for FB1 and FB2; 100, 250, and 500 ng/g for FB3) for accuracy and precision. Recoveries of FB1 ranged from 93 to 98% with RSD values of 3-8%. Recoveries of FB2 ranged from 104 to 108%, with RSD values of 2-6%. Recoveries of FB3 ranged from 94 to 108%, with RSD values of 2-5%.