Applications of CE SDS gel in development of biopharmaceutical antibody-based products

CE SDS gel technique offers many advantages over the traditional labor-intensive SDS PAGE slab gel technology. The CE-based method has increasingly been applied to many protein analysis applications. Specific examples are provided for monoclonal antibody (mAb), though the technique can be adapted to many other therapeutic protein products. Applications of CE SDS gel method using Beckman PA800 with UV detection are presented and discussed with respect to mAb analysis, such as purity, quantitation of non-glycosylated heavy chain (NGHC) peak, identity, and stability. The stability of mAb is evaluated with respect to formulation buffer, accelerated temperature stress, UV light-exposure, and high pH conditions. Both reducing and non-reducing CE SDS gel conditions were applied and optimized to characterize mAb products. The data presented provides a "taste" of what CE SDS gel method can do to support the development of mAb products from early clone screening for product quality to the final product characterization. Since the CE SDS gel method is automatable, quantitative, robust, and allows for relatively high throughput, it provides both great analytical capacity and product coverage for a wide spectrum of protein product development in biopharmaceutical industry.     

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.     

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.     

Optimization of CE-SDS Method for Antibody Separation Based on Multi-Users Experimental Practices

Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS) method with UV detection was developed and satisfactorily used for determination of purity and manufacturing consistency of a monoclonal antibody (MAb) at Amgen Inc. (Seattle, WA). When this method was applied to some other MAbs, several problems with method robustness became apparent. These issues resulted in abnormal Electropherogram (e-gram) profiles potentially linked to various parameters specific molecules analyzed, sample formulation buffer composition, CE-SDS gel matrix type, and operators. A multi-users interest group (called CE Users Forum) was formed to systematically investigate and understand these issues. The CE Users Forum first identified the issues which needed resolution, defined group experiments to better understand the problem and to test potential solutions, and together defined a generic (platform) CE-SDS method for MAbs. Two CE instruments, Agilent HP^3DCE and Beckman PA 800, two CE-SDS gel matrices, BioRad and Beckman gels, as well as different types of MAbs in various buffers were used in this investigation. We present here a platform CE-SDS method for purity determination of MAbs. Method optimization and trouble-shooting procedures by the CE Users Forum played a key role in delivering a robust analytical method for characterization of antibodies by improving instrumental and experimental parameters such as instrument variability, instrument operating parameters, operator training, and reagent stability. The optimized CE-SDS method is used during process development and has been transferred to the quality control (QC) lab as a purity assay for lot release testing of therapeutic antibodies. Any trained analyst can successfully perform this method. A group such as the CE Users Forum is a good way to integrate best practices and solve technical issues in a cooperative environment.Presented at: 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     

Elimination of light chain tailing in reducing capillary electrophoresis with sodium dodecyl sulfate analysis of a monoclonal antibody

Capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) is a common analytical technique for investigating the purity and molecular size heterogeneity of monoclonal antibody (mAb) drugs. In reducing CE-SDS analysis of mAb-A, the light chain (LC) peak exhibited severe tailing, seriously affecting the purity analysis. The purposes of this investigation are to clarify the source of tailing and develop a more appropriate CE-SDS method to eliminate LC tailing. The degree of LC tailing was closely related to the mAb concentration, SDS concentration, and injection amount, and more hydrophobic detergents, such as sodium hexadecyl sulfate (SHS) and sodium tetradecyl sulfate (STS), could be used instead of SDS to obtain better peak shapes. The results also indicated that the tailing was caused by the binding problem associated with SDS, and SHS/STS could provide a more stable and uniform complexation for the LC. In summary, the method we developed successfully eliminated the LC tailing and provided a robust characterization of mAb-A in reducing CE-SDS analysis.     

Quantitative impurity analysis of monoclonal antibody size heterogeneity by CE-LIF: example of development and validation through a quality-by-design framework

This paper describes the framework of quality by design applied to the development, optimization and validation of a sensitive capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) assay for monitoring impurities that potentially impact drug efficacy or patient safety produced in the manufacture of therapeutic MAb products. Drug substance or drug product samples are derivatized with fluorogenic 3-(2-furoyl)quinoline-2-carboxaldehyde and nucleophilic cyanide before separation by CE-SDS coupled to LIF detection. Three design-of-experiments enabled critical labeling parameters to meet method requirements for detecting minor impurities while building precision and robustness into the assay during development. The screening design predicted optimal conditions to control labeling artifacts while two full factorial designs demonstrated method robustness through control of temperature and cyanide parameters within the normal operating range. Subsequent validation according to the guidelines of the International Committee of Harmonization showed the CE-SDS/LIF assay was specific, accurate, and precise (RSD ≤ 0.8%) for relative peak distribution and linear (R > 0.997) between the range of 0.5-1.5 mg/mL with LOD and LOQ of 10 ng/mL and 35 ng/mL, respectively. Validation confirmed the system suitability criteria used as a level of control to ensure reliable method performance.     

Four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis

Vaccines against infectious diseases are urgently needed. Therefore, modern analytical method development should be as efficient as possible to speed up vaccine development. The objectives of the study were to identify critical method parameters (CMPs) and to establish a set of steps to efficiently develop and validate a CE-SDS method for vaccine protein analysis based on a commercially available gel buffer. The CMPs were obtained from reviewing the literature and testing the effects of gel buffer dilution. A four-step approach, including two multivariate DoE (design of experiments) steps, was proposed, based on CMPs and was verified by CE-SDS method development for: (i) the determination of influenza group 1 mini-hemagglutinin glycoprotein; and (ii) the determination of polio virus particle proteins from an inactivated polio vaccine (IPV). The CMPs for sample preparation were incubation temperature(s) and time(s), pH, and reagent(s) concentration(s), and the detection wavelength. The effects of gel buffer dilution revealed the CMPs for CE-SDS separation to be the effective length, the gel buffer concentration, and the capillary temperature. The four-step approach based on the CMPs was efficient for the development of the two CE methods. A four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis was successfully established.     

Development,validation, and implementation of capillary gel electrophoresis as a replacement for SDS‐PAGE for purity analysis of IgG2 mAbs

Capillary gel electrophoresis (CGE) methods with UV detection were developed for reduced and non‐reduced mAb analysis. These methods can be used to evaluate mAb purity, offering more reproducible quantitation compared with that of traditional SDS‐PAGE methods. These CGE methods have been utilized as platform technology for bioprocess development, formulation development, mAb characterization, drug substance/drug product release testing as well as a required methodology for stability testing. We have found these CGE methods to be applicable across a platform of mAbs in preclinical and clinical development, with the majority of mAbs requiring no modification to the method conditions. This methodology has been ICH validated and transferred to several supporting organizations. The data presented herein describes the development of CGE methodology, platform application to mAb purity analysis, ICH validation, reliability metrics, and considerations on technology enhancement for improved performance and throughput.     

Sixteen capillary electrophoresis applications for viral vaccine analysis

A broad range of CE applications from our organization is reviewed to give a flavor of the use of CE within the field of vaccine analyses. Applicability of CE for viral vaccine characterization, and release and stability testing of seasonal influenza virosomal vaccines, universal subunit influenza vaccines, Sabin inactivated polio vaccines (sIPV), and adenovirus vector vaccines were demonstrated. Diverse CZE, CE-SDS, CGE, and cIEF methods were developed, validated, and applied for virus, protein, posttranslational modifications, DNA, and excipient concentration determinations, as well as for the integrity and composition verifications, and identity testing (e.g., CZE for intact virus particles, CE-SDS application for hemagglutinin quantification and influenza strain identification, chloride or bromide determination in process samples). Results were supported by other methods such as RP-HPLC, dynamic light scattering (DLS), and zeta potential measurements. Overall, 16 CE methods are presented that were developed and applied, comprising six adenovirus methods, five viral protein methods, and methods for antibodies determination of glycans, host cell-DNA, excipient chloride, and process impurity bromide. These methods were applied to support in-process control, release, stability, process- and product characterization and development, and critical reagent testing. Thirteen methods were validated. Intact virus particles were analyzed at concentrations as low as 0.8 pmol/L. Overall, CE took viral vaccine testing beyond what was previously possible, improved process and product understanding, and, in total, safety, efficacy, and quality.     

Combination of SDS‐PAGE and intact mass analysis for rapid determination of heterogeneities in monoclonal antibody therapeutics

mAbs are currently mainstream in biopharmaceuticals, and their market has been growing due to their high target specificity. Characterization of heterogeneities in mAbs is performed to secure their quality and safety by physicochemical analyses. However, they require time‐consuming task, which often strain the resources of drug development in pharmaceuticals. Rapid and direct method to determine the heterogeneities should be a powerful tool for pharmaceutical analysis. Considering the advantages of electrophoresis and MS, this study addresses the combination of SDS‐PAGE and intact mass analysis, which provides direct, rapid, and orthogonal determination of heterogeneities in mAb therapeutics. mAb therapeutics that migrated in SDS‐PAGE were recovered from gel by treatment with SDC‐containing buffer. Usage of SDC‐containing buffer as extraction solvent and ethanol‐based staining solution enhanced the recovery of intact IgG from SDS‐PAGE gels. Recovery of mAbs reached more than 86% with 0.2% SD. The heterogeneities, especially N ‐glycan variants in the recovered mAb therapeutics, were clearly determined by intact mass analysis. We believe that the study is important in pharmaceuticals‧ perspective since orthogonal combination of gel electrophoresis and intact mass analysis should be pivotal role for rapid and precise characterization of mAbs.     

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.     

Microchip electrophoresis separation coupled to mass spectrometry (MCE–MS) for the rapid monitoring of multiple quality attributes of monoclonal antibodies

Therapeutic monoclonal antibodies (mAbs) are highly heterogeneous as a result of posttranslational modifications (PTMs) during bioprocessing and storage. The modifications that impact mAb product quality are regarded as critical quality attributes and require monitoring. The conventional LC–mass spectrometer (MS) method used for product quality monitoring may require protein A purification prior to analysis. In this paper, we present a high-throughput microchip electrophoresis (<4 min) in-line with MS (MCE–MS) that enables baseline separation and characterization of Fc, Fd′, and light chain (LC) domains of IdeS-treated mAb sample directly from bioreactor. The NISTmAb was used to optimize the MCE separation and to assess its capability of multiple attribute monitoring. The MCE–MS can uniquely separate and characterize deamidated species at domain level compared to LC–MS method. Two case studies were followed to demonstrate the method capability of monitoring product quality of mAb samples from stability studies or directly from bioreactors.     

Applications of imaged capillary isoelectric focussing technique in development of biopharmaceutical glycoprotein-based products

CE-based methods have increasingly been applied to the analysis of a variety of different type proteins. One of those techniques is imaged capillary isoelectric focusing (icIEF), a method that has been used extensively in the field of protein-based drug development as a tool for product identification, stability monitoring, and characterization. It offers many advantages over the traditional labor-intensive IEF slab gel method and even standard cIEF with on-line detection technologies with regard to method development, reproducibility, robustness, and speed. Here, specific examples are provided for biopharmaceutical glycoprotein products such as mAbs, erythropoietin (EPO), and recombinant Fc-fusion proteins, though the technique can be adapted for many other therapeutic proteins. Applications of iCIEF using a Convergent Bioscience instrument (Toronto, Canada) with whole-field imaging technology are presented and discussed. These include a quick method to establish an identity test for many protein-based products, product release, and stability evaluation of glycoproteins with respect to charge heterogeneity under accelerated temperature stress, different pH conditions, and in different formulations. Finally, characterization of glycoproteins using this iCIEF technology is discussed with respect to biosimilar development, clone selection, and antigen binding. The data presented provide a "taste' of what icIEF method can do to support the development of biopharmaceutical glycoprotein products from early clone screening for better product candidates to characterization of the final commercial products.     

Aspects of surface modification, structure characterization, thermal stability and metal selectivity properties of silica gel phases-immobilized-amine derivatives

Four silica gel phases-bound-amine derivatives (I-IV) were prepared based on chemical immobilization technique. The surface modification was identified by determination of the coverage values in mmol g⁻¹ via thermal desorption method (1.463-1.807) and elemental analysis of nitrogen and carbon contents (1.089-2.456). Structure characterization related to immobilization of the amine derivatives was accomplished and evaluated by means of infrared (IR) and secondary ion mass spectrometric (SIMS) technique. The modified silica gel phases (I-IV) along with their interaction products with copper(II) were also examined by electron impact mass spectrometric analysis (EI-MS) as a method for evaluation of their thermal stability and structure elucidation. Potentiometric titration as a method of characterization was applied for the modified silica gel phases (II-IV) and their copper(II)-adduct. A series of bi- and trivalent metal ions were selected to focus more aspects of the selectivity properties incorporated into the modified silica gel phases for binding and interaction with these metals based on determination of the distribution coefficient and separation factor. The results of these evaluation processes were found to prove higher selectivity and preference of these four phases for binding with lead(II) and cadmium(II) compared to other metal ions.     

Minimization of artifact protein aggregation using tetradecyl sulfate and hexadecyl sulfate in capillary gel electrophoresis under reducing conditions

In the biopharmaceutical industry, CE-SDS assesses the purity, heterogeneity, and stability of therapeutic proteins. However, for mAb-1 and mAb-2, typical CE-SDS under reducing conditions produced atypical protein peak profiles, which led to biased purity results, thus were not acceptable for biologics manufacturing. This bias was caused by the formation of method-induced higher molecular weight artifacts, the levels of which correlated with protein concentration. Here we show that adding sodium tetradecyl and hexadecyl sulfates to the sample and the sieving gel buffer solutions was required to prevent formation of aggregate artifacts and to maintain detergent:protein uniformity, suggesting their importance during the sample preparation steps of heat denaturation and subsequent cooling as well as during capillary migration. For these proteins, we show that this uniformity was likely due to the ability of these detergents to bind proteins with markedly higher affinities compared to SDS. “CE-SC_XS” methods (where CE-SC_XS is CGE using detergent composed of a sodium sulfate head group and a hydrocarbon tail, with “C_X” representing various tail lengths), were developed with a sodium tetradecyl sulfate sample buffer and a sodium hexadecyl sulfate containing sieving gel buffer that minimized artifacts and provided robust characterization and release results for mAb-1 and mAb-2.     

聚N-异丙基丙烯酰胺硅胶键合固定相的制备与评价

以3-巯丙基三甲氧基硅烷为偶联剂,将聚N-异丙基丙烯酰胺(PNIPAM)键合到硅胶上,制得了键合固定相（SI-PNIPAM）填料,并用元素分析、红外光谱等对其进行了表征。以甲醇-水为二元流动相,用多环芳烃、碱性物质对该固定相进行了色谱评价,并考察了该固定相的适用pH范围及水解稳定性。结果表明:该固定相具有较好的色谱性能与温敏特性,并且在pH 2.5～7.5时稳定性良好。     

Immunoglobulin dynamics, conformational fluctuations, and nonlinear elasticity and their effects on stability

The relationships between protein dynamics, function, and stability are incompletely understood. Two external perturbations (temperature and pH) were used to modulate the flexibility and stability of an IgG1kappa monoclonal antibody (mAb) in an attempt to better understand the possible correlations between flexibility and stability. Ultrasonic velocimetry, densitometry, differential scanning calorimetry (DSC), and pressure perturbation calorimetry (PPC) were used to experimentally determine the adiabatic and isothermal compressibility, expansibility, fractional volumes of unfolding, and various nonlinear thermoacoustical parameters as a function of pH and temperature. By combining these results, state parameter fluctuations were calculated from fundamental statistical mechanical relationships. The most dynamic and rigid mAb ensemble is measured at pH 4 and 6, respectively, based on state parameter fluctuations and compressibility. The effect of pH appears to couple mAb dynamics to solvent fluctuations, which control its dynamics and stability. A nonlinear response to mechanical perturbation, comparable to that seen with many polymers, is observed for this monoclonal antibody at pH 4-8. This behavior is characterized as strongly anisotropic and anharmonic, especially at pH 4. The midpoint of thermal unfolding as measured by DSC does not necessarily correlate with flexibility.     

Development and Validation of Salt Gradient CEX Chromatography Method for Charge Variants Separation and Quantitative Analysis of the IgG mAb-Cetuximab

Ion exchange chromatography is widely used for charge variant analysis of proteins, including monoclonal antibodies. In this study, a simple and robust salt gradient cation exchange chromatography was developed and validated for quantitative determination of cetuximab in biopharmaceutical formulations. For this purpose, we investigated the effect of various parameters including buffer composition, column temperature, pH, gradient volume and flow rate on chromatographic separation of charge variants to achieve the acceptable peak separation, and the optimum condition was selected. Validation of the method was done in accordance with the International Conference on Harmonization (ICH) guidelines. The developed method was found to provide a linear regression over the concentration range of 0.06–2.00 mg mL^?1 yielding a correlation coefficient of 0.9972. The limits of detection and quantification for the developed method were 0.02 and 0.06 mg mL^?1, respectively. The intra-day and inter-day precision had relative standard deviation values?≤?2.7%. The robustness of the method was assessed by changes in the applied pH range of buffer, temperature, mobile phase composition, and flow rate. Specificity of the method was confirmed by evaluation of baseline resolution of the mAb variants from product excipients, which showed no interference between excipients and cetuximab. The stability indicating capability of this method was determined using photodegraded, and mechanically and thermally stressed samples. The proposed method could be applied as a simple, precise, and robust quantitative technique which can be reproduced in any labs for the high-throughput quality control and stability assessment of in-process and final product samples.     

The synthesis of poly(hydroxamic acid) from poly(acrylamide)

Poly(hydroxamic acid) in gel or water soluble from was prepared from the reaction of poly(acrylamide) and hydroxylamine in basic aqueous solution (pH > 12) at room temperature. The polymers were composed of 70% hydroxamic acid groups, less than 5% carboxylic acid groups, and 25% unreacted amide groups. The polymers exhibited high affinity to iron(III) and copper(II) in the pH range of 1 to 5 with a high binding rate. A binding of 3 mmol/g for both metals was achieved. Preliminary tests demonstrated the urease inhibitory activity of both linear and crosslinked poly(hydroxamic acids).     

Degradation performance of polyglutamic acid and its application of calcium supplement

Degradable biopolymers with functional groups play a crucial role in the biomedical field. In this case, the influences of temperature and pH values on the degradation performance of poly (γ‐glutamic acid) (γ‐PGA) were fully explored by gel permeation chromatography. Further, γ‐PGA‐Ca was prepared by using calcium chloride to react with the low molecular weight γ‐PGA and characterized by Fourier transform infrared, differential scanning calorimetry test, gel permeation chromatography, atomic absorption spectrophotometric, Ca²⁺ release in vivo, and cytotoxicity experiments. Furthermore, Caco‐2 cell model was constructed to study the mechanism of γ‐PGA‐Ca intestinal absorption. Results indicated that low pH value and high temperature are the suitable conditions for the degradation of γ‐PGA. It also suggested that γ‐PGA‐Ca can be used as calcium supplements due to its high rate of absorption.