Intercompany Study to Evaluate the Robustness of Capillary Isoelectric Focusing Technology for the Analysis of Monoclonal Antibodies

Interlaboratory comparisons are essential to bringing emerging technologies into biopharmaceutical industry practice and regulatory acceptance. As a result, an international team including 12 laboratories from 10 independent biopharmaceutical companies in the United States and Switzerland was formed to evaluate the precision and robustness of capillary isoelectric focusing (CIEF) to assess the charge heterogeneity of monoclonal antibodies. The different laboratories determined the apparent pI and the relative distribution of the charge isoforms of a representative monoclonal antibody (rMAb) sample using the same CIEF method. Statistical evaluation of the data was performed to determine within and between-laboratory consistencies and outlying information. The apparent pI data generated for each charge variant peak showed very good precision between laboratories with percentage of RSD values of ??0.5%. Similarly, the percentage of RSD for the rMAb charge variants percent peak area values are ??4.4% across different laboratories with different analysts using different lots of ampholytes and multiple instruments. Taken together, these results validate the appropriate use of CIEF in the biopharmaceutical industry in support of regulatory submissions.     

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.     

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     

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.     

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.

     

A comparative study of CE-SDS,SDS-PAGE,and Simple Western—Precision,repeatability, and apparent molecular mass shifts by glycosylation

SDS gel electrophoresis is a commonly used approach for monitoring purity and apparent molecular mass (Mr) of proteins, especially in the field of quality control of biopharmaceutical proteins. The technological installation of CE-SDS as the replacement of the slab gel technique (SDS-PAGE) is still in progress, leading to a continuous improvement of CE-SDS instruments. Various CE-SDS instruments, namely Maurice (CE-SDS/CE-SDS PLUS) and Wes by ProteinSimple as well as the microchip gel electrophoresis system LabChip® GXII Touch™ HT by PerkinElmer were tested for precision and repeatability compared to SDS-PAGE (Bio-Rad). For assessing these quality control parameters, standard model proteins with minor post-translational modifications were used. Overall, it can be concluded that the CE-SDS-based methods are similar to SDS-PAGE with respect to these parameters. Quality characteristics of test systems gain more significance by testing proteins that do not behave like model proteins. Therefore, glycosylated proteins were analyzed to comparatively investigate the influence of glycosylation on Mr determination in the different instruments. In some cases, high deviations were found both among the methods and with regard to reference values. This article provides possible explanations for these findings.     

Radionuclide traceability for U.S. Department of Energy Environmental Management Radioanalytical Services

In 1999, the Department of Energy Office of Environmental Management (DOE-EM) National Analytical Management Program (NAMP) established a Radiological Traceability Program (RTP) as a new initiative for the radioanalytical acitivies related to the environmental programs conducted throughout the DOE complex. The National Analytical Management Program entered into an interagency agreement with the National Institute of Standards and Technology (NIST) to establish traceability to the national standard for DOE-EM radioanalytical activities through the NIST/reference laboratory concept as described in ANSI N42.23-1996.¹ Using the criteria established by the RTP, NAMP named two DOE-EM laboratories as reference or secondary laboratories and established a program with NIST that demonstrated the concept of traceability. In order to gain and maintain traceability to NIST, each reference laboratory must meet the performance criteria as defined by the RTP and NAMP. Traceability to NIST is tiered down to each radioanalytical laboratory (monitor or service) that successfully participates in the performance-evaluation programs offered by the reference laboratories. Essential to the RTP is the demonstration that the reference laboratories can produce performance-testing (PT) materials of high quality as well as analyze/verify the radionuclide concentration to the required accuracy and precision. This paper presents the elements of the RTP and the program requirements of NIST and the reference laboratories.     

Improved CE(SDS)-CZE-MS method utilizing an 8-port nanoliter valve

Capillary sieving electrophoresis utilizing SDS (CE(SDS)) is one of the most applied methods for the analysis of antibody (mAb) size heterogeneity in the biopharmaceutical industry. Inadequate peak identification of observed protein fragments is still a major issue. In a recent publication, we introduced an electrophoretic 2D system, enabling online mass spectrometric detection of generic CE(SDS) separated peaks and identification of several mAb fragments. However, an improvement regarding system stability and handling of the approach was desired. Here, we introduce a novel 8-port valve in conjunction with an optimized decomplexation strategy. The valve contains four sample loops with increased distances between the separation dimensions. Thus, successively coinjection of solvent and cationic surfactant without any additional detector in the second dimension is enabled, simplifying the decomplexation strategy. Removal efficiency was optimized by testing different volumes of solvents as presample and cationic surfactant as postsample zone. 2D measurements of the light and heavy chain of the reduced NIST mAb with the 8-port valve and the optimized decomplexation strategy demonstrates the increased robustness of the system. The presented novel set-up is a step toward routine application of CE(SDS)-CZE-MS for impurity characterization of proteins in the biopharmaceutical field.     

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.     

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.     

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.     

Determination of vitamin K in milk and infant formulas by liquid chromatography: collaborative study

A simple procedure for determination of vitamin K1 was developed for routine compliance monitoring of supplemented infant formula and measurement of endogenous levels in milk and milk powders. Samples are digested with lipase and extracted into hexane; and aliquot is evaporated, reconstituted in methanol, and analyzed by reversed-phase LC. Post-column zinc reduction of phylloquinone facilitates detection by fluorescence. The procedure was subjected to an AOAC collaborative study involving 8 materials, each in blind duplicate, across the range of 5-120 micrograms/100 g solids and including NIST 1846 reference material. Thirty-three laboratories returned valid data which were then statistically analyzed for outliers and precision parameters. Mean RSDR (%) was 6.53 (4.33-10.94), with a mean HORRAT value of 0.33 (0.23-0.43) and RSDr:RSDR ratio of 0.74. K1 isomers (cis and trans) were aggregated with conventional C18 columns, but may be selectively estimated with use of the C30 column.     

Preparation and value assignment of standard reference material 968e fat-soluble vitamins, carotenoids, and cholesterol in human serum

Standard Reference Material 968e Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum provides certified values for total retinol, γ- and α-tocopherol, total lutein, total zeaxanthin, total β-cryptoxanthin, total β-carotene, 25-hydroxyvitamin D₃, and cholesterol. Reference and information values are also reported for nine additional compounds including total α-cryptoxanthin, trans- and total lycopene, total α-carotene, trans-β-carotene, and coenzyme Q₁₀. The certified values for the fat-soluble vitamins and carotenoids in SRM 968e were based on the agreement of results from the means of two liquid chromatographic methods used at the National Institute of Standards and Technology (NIST) and from the median of results of an interlaboratory comparison exercise among institutions that participate in the NIST Micronutrients Measurement Quality Assurance Program. The assigned values for cholesterol and 25-hydroxyvitamin D₃ in the SRM are the means of results obtained using the NIST reference method based upon gas chromatography-isotope dilution mass spectrometry and liquid chromatography-isotope dilution tandem mass spectrometry, respectively. SRM 968e is currently one of two available health-related NIST reference materials with concentration values assigned for selected fat-soluble vitamins, carotenoids, and cholesterol in human serum matrix. This SRM is used extensively by laboratories worldwide primarily to validate methods for determining these analytes in human serum and plasma and for assigning values to in-house control materials. The value assignment of the analytes in this SRM will help support measurement accuracy and traceability for laboratories performing health-related measurements in the clinical and nutritional communities.     

Results of the ASTM Nuclear Methods intercomparison on NIST apple and peack leaves standard reference materials

The ASTM Task Group on Nuclear Methods of Chemical Analysis (E10.05.12) has conducted a trace element intercomparison among some of its members over the past two years. Eight non-NIST laboratories submitted data using nuclear techniques, with a total of 111 values for the apple leaves and 116 values for the peach leaves, on 46 and 50 elements, respectively. This intercomparison provided a unique opportunity for the analytical laboratories, because the analytical values submitted could be later compared to the NIST certified values. For the seven elements which were certified by NIST and had three or more intercomparison values, the results showed that: 1) 61% of all 56 intercomparison values submitted had results whose stated uncertainty overlapped the uncertainty limits of the NIST certified values, and 2) less than 6% of the intercomparison values had means which fell outside ±20% of the NIST values. In general, the intercomparison values submitted showed excellent agreement with the NIST values. However, many reported uncertainties accompanying intercomparison values appeared overly optimistic.     

The development and inter-laboratory verification of LC-MS libraries for organic chemicals of environmental concern

The development, verification, and comparison study between LC-MS libraries for two manufacturers’ instruments and a verified protocol are discussed. Compounds in the libraries are among those considered by the U.S. EPA Office of Water as threats to drinking water including pesticides, drugs of abuse, and pharmaceuticals. The LC-MS library protocol was verified through an inter-laboratory study that involved Federal, State, and private laboratories. The results demonstrated that the libraries are transferable between the same manufacturer's product line, and have applicability between manufacturers. Although ion abundance ratios within mass spectra were shown to be different between the manufacturers’ instruments, the NIST search engine match probability was at 96% or greater for 64 out of 67 compounds evaluated.     

Recent developments in food-matrix Reference Materials at NIST

Since 1996, the National Institute of Standards and Technology (NIST) has developed several food-matrix Standard Reference Materials (SRMs) characterized for nutrient concentrations. These include SRM 1544 Fatty Acids and Cholesterol in a Frozen Diet Composite, SRM 1546 Meat Homogenate, SRM 1548a Typical Diet, SRM 1566b Oyster Tissue, SRM 1846 Infant Formula, and SRM 2383 Baby Food Composite. Three additional materials--SRM 1946 Lake Superior Fish Tissue, SRM 2384 Baking Chocolate, and SRM 2385 Spinach--are in preparation. NIST also recently assigned values for proximate (fat, protein, etc.), individual fatty acid, and total dietary fiber concentrations in a number of existing SRMs and reference materials (RMs) that previously had values assigned for their elemental composition. NIST has used several modes for assignment of analyte concentrations in the food-matrix RMs, including the use of data provided by collaborating laboratories, alone and in combination with NIST data. The use of data provided by collaborating food industry and contract laboratories for the analysis of food-matrix RMs has enabled NIST to provide assigned values for many analytes that NIST does not have the resources or analytical expertise to measure.     

web-based interactive data processing: application to stable isotope metrology

To address a fundamental need in stable isotope metrology, the National Institute of Standards and Technology (NIST) has established a web-based interactive data-processing system accessible through a common gateway interface (CGI) program on the internet site http://www. nist.gov/widps-co2. This is the first application of a web-based tool that improves the measurement traceability afforded by a series of NIST standard materials. Specifically, this tool promotes the proper usage of isotope reference materials (RMs) and improves the quality of reported data from extensive measurement networks. Through the International Atomic Energy Agency (IAEA), we have defined standard procedures for stable isotope measurement and data-processing, and have determined and applied consistent reference values for selected NIST and IAEA isotope RMs. Measurement data of samples and RMs are entered into specified fields on the web-based form. These data are submitted through the CGI program on a NIST Web server, where appropriate calculations are performed and results returned to the client. Several international laboratories have independently verified the accuracy of the procedures and algorithm for measurements of naturally occurring carbon-13 and oxygen-18 abundances and slightly enriched compositions up to approximately 150% relative to natural abundances. To conserve the use of the NIST RMs, users may determine value assignments for a secondary standard to be used in routine analysis. Users may also wish to validate proprietary algorithms embedded in their laboratory instrumentation, or specify the values of fundamental variables that are usually fixed in reduction algorithms to see the effect on the calculations. The results returned from the web-based tool are limited in quality only by the measurements themselves, and further value may be realized through the normalization function. When combined with stringent measurement protocols, two- to threefold improvements have been realized in the reproducibility of carbon-13 and oxygen-18 determinations across laboratories.     

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.     

Towards standardization of external quality assessment schemes by using bias values based on biological variation

The precision and trueness of current instruments and methods in clinical laboratories is much better than in the past. However, the z-score and other comparison variables that are currently used in external quality assessment programs are based on relative data. Thus, they may change from program to program and also change over time within the same program and consequently may not be useful or cost-effective. We therefore devised a test-specific decision limit for accepting or rejecting test results based on a combination of the data of within- and between-subject biological variations. We then applied these limits to a group of tests performed in our laboratory and compared our results with those of external quality assessment programs. In addition, we combined external and internal quality control data on the same graph and prepared a two-dimensional graph for different levels of control sera. Inspection of all results of control sera on this new graph was more useful for decision making. We concluded that the z-score is not reliable for comparisons of test results in external quality assessment. As a substitute for this currently accepted practice, we assert that control limits based on biological variation are more reliable, and can be useful in the evaluation of both external and internal quality assessments and their combination on this new graph.     

Breakfast cereal sampling study for nutritional elements

The National Institute of Standards and Technology (NIST) has established a Dietary Supplement Laboratory Quality Assurance Program (DSQAP) in collaboration with the National Institutes of Health Office of Dietary Supplements (NIH-ODS). The DSQAP invites laboratories twice annually to participate in interlaboratory studies where participants elect to measure concentrations of nutritional and/or toxic elements as well as active and/or marker compounds. One of these studies was designed to determine the effects of material granularity and sample processing techniques on measurement variability (precision) as well as to provide participating laboratories information on their performance relative to the NIST assigned values (bias) and to the other participants (concordance). Participants were asked to determine the mass fractions of Ca, Fe, and Zn, in mg/kg, in six breakfast cereal samples. Cereal samples consisted of three ground materials (homogenized wheat, wheat, and rice), two flake materials (wheat and rice) and a partially crushed material (a wheat/rice mixture). In general, approximately 25 % of the laboratories processed and analyzed the suite of six cereal materials with adequate to exemplary measurement precision. Over half of the laboratories (60 %) experienced measurement issues related to only a particular type of cereal matrix or for only a single element. A small number (15 %) of laboratories experienced significant sample processing or measurement problems. Future studies planned by the DSQAP may be designed to use commercial products to aid laboratories with their sampling and analytical techniques.