Intact and middle-down CIEF of commercial therapeutic monoclonal antibody products under non-denaturing conditions

A two-step CIEF with chemical mobilization was developed for charge profiling of the therapeutic mAb rituximab under non-denaturing separation conditions. CIEF of the intact mAb was combined with a middle-down approach analyzing Fc/2 and F(ab´)₂ fragments after digest with a commercial cysteine protease (IdeS). CIEF methods were optimized separately for the intact mAb and its fragments due to their divergent pIs. Best resolution was achieved by combining Pharmalyte (PL) 8–10.5 with PL 3–10 for variants of intact rituximab and of F(ab´)₂ fragments, respectively, whereas PL 6.7–7.7 in combination with PL 3–10 was used for Fc/2 variants. Charge heterogeneity in Fc/2 dominates over F(ab´)₂. In addition, a copy product of rituximab, and adalimumab were analyzed. Both mAbs contain additional alkaline C-terminal lysine variants as confirmed by digest with carboxypeptidase B. The optimized CIEF methods for intact mAb and Fc/2 were tested for their potential as platform approaches for these mAbs. The CIEF method for Fc/2 was slightly adapted in this process. The pI values for major intact mAb variants were determined by adjacent pI markers resulting in 9.29 (rituximab) and 8.42 (adalimumab). In total, seven to eight charge variants could be distinguished for intact adalimumab and rituximab, respectively.     

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.     

Protocol of capillary isoelectric focusing to separate extremely acidic and basic proteins

A new set-up was constructed for capillary isoelectric focusing (CIEF) involving a sampling capillary as a bypass fixed to the separation capillary. Sample solutions were subjected to a previously established pH gradient from the sample capillary. Besides performing conventional CIEF, the separation of ampholytic compounds with isoelectric points (p/s) beyond the pH gradient was carried out on this system. This method was termed as pH gradient driven electrophoresis (PGDE) and the basic mathematical expressions were derived to express the dynamic fundamentals. Proteins such as lysozyme, cytochrome C, and pepsin with p/s higher than 10 or below 3 were separated in a pH gradient provided by Pharmalyte (pH 3-10). Finally, this protocol convincingly exhibited its potential in the separation of a solution of chicken egg white.     

Analysis of four therapeutic monoclonal antibodies by online capillary isoelectric focusing directly coupled to quadrupole time‐of‐flight mass spectrometry

Capillary isoelectric focusing (cIEF) was online coupled to a Q‐TOF MS by a flow‐through microvial interface for the analysis of therapeutic mAb. Intact molecular weights obtained from the mass spectrum deconvolution of separated charge variants provided information on the structural heterogeneity of therapeutic mAbs. A sandwich cIEF–MS configuration composed of anolyte, sample, and catholyte segments sequentially injected into a neutrally coated capillary was used for the charge heterogeneity separation of four mAbs. Acetic acid and ammonium hydroxide were used in places of the non‐volatile acids and bases commonly used for IEF but are incompatible with online MS detection. Glycerol was added as the anti‐convective reagent. A chemical modifier was mixed with the cIEF effluent in the flow‐throw microvial to maintain the ESI stability and to mitigate ion suppression from the co‐eluted carrier ampholytes and glycerol. Analysis of mAb samples have shown relative populations of two basic variants originating from C‐terminal lysine process and acidic variant of deamidation. The lysine clippings, deamidation, and sialic acid modification in oligosaccharide chains were revealed in infliximab. Two lysine clipping variants and a deamidated variant were observed in adalimumab. The duplicate analyses of a reference mAb demonstrated five charge variants separated by cIEF due to some unidentified modifications, as their mass spectra shared close similarities. The mAb analyses demonstrated the feasibility of the cIEF–MS method, and they demonstrated how charge and structural variants and minor differences in therapeutic mAbs are observed with this technology. Online cIEF–MS is an information rich technology with high throughput, demonstrated by the initial data presented here.     

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.     

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 analysis of charge variants of monoclonal antibodies with capillary zone electrophoresis in dynamically coated fused-silica capillary

A capillary zone electrophoresis (CZE) method was developed for the rapid analysis of charge heterogeneity of immunoglobulin G (IgG) monoclonal antibodies (mAbs). The separation was carried out in a short, dynamically coated fused-silica capillary. A number of separation parameters were investigated and optimized, including pH, concentration of the separation buffer (ε-amino caproic acid), concentration of the triethylenetetramine (TETA) dynamic coating, the capillary internal diameter and the field strength used for the separation. The effects of between-run flushing of the capillary and the data acquisition rate were also evaluated. Under the optimized conditions, a fast (<5 min), selective and reproducible separation of mAb charge variants was achieved under a very high electric field strength (1000 V/cm). This method also requires only a short conditioning of the capillary, with between-run conditioning completed within 2 min. The method was evaluated for specificity, sensitivity, linearity, accuracy and precision. The same separation conditions were applied to the rapid separation (2-5 min) of charge variants of multiple monoclonal antibodies with pI in the range of 7.0-9.5. Compared with other existing methods for charge variants analysis, this method has several advantages including a short run time, rapid capillary conditioning and simple sample preparation.     

Optimization of an IgG1 CIEF separation by using narrow-range ampholytes and DMSO as protein solubilizer

CIEF is a powerful separation tool utilized in the characterization and relative quantitation of therapeutic mAb charged isoforms. However, one CIEF method is not capable of separating all mAbs with high resolution and reproducibility. Optimization of sample composition and separation parameters is expected when developing a CIEF method for a specific mAb. This paper summarizes a root cause investigation into why a validated CIEF separation method for MAK33 (a type of IgG1) was no longer reproducible. In addition, this paper introduces the concept of sample focusing volume, which is defined as the actual capillary volume occupied by the sample after focusing and explains why there is less protein precipitation and aggregation when using narrow-range ampholytes than broad-range ampholytes. The use of DMSO as protein solubilizer and possible replacement of urea is also explored in this work. Finally, this paper demonstrates that a new optimized CIEF method can achieve over 100 reproducible high-resolution separations of MAK33 per neutral-coated capillary.     

Separation of human cerebrospinal fluid proteins by capillary isoelectric focusing in the absence of denaturing agents

Conditions of capillary isoelectric focusing (CIEF) to separate human cerebrospinal fluid (CSF) proteins were examined referring to those which we have established for the separation of human plasma/serum proteins. Since the average protein concentration in CSF is about 1/200 of plasma and the salt concentration is at almost the same level as plasma, desalting of CSF samples with minimum dilution was a prerequisite for CIEF analysis of CSF proteins. We constructed an apparatus to dialyze CSF at the level of 20-30 microL, this volume being sufficient for 3-4 repeated analyses of the CSF sample. To trace the process of dialysis, a simple device to measure the conductivity of the dialyzate was also constructed. Most of the CIEF conditions for plasma protein analysis could be applied for CSF protein analysis. However, the addition of N,N,N',N'-tetramethylethylenediamine (TEMED) at a suitable concentration was necessary to improve the resolution of basic proteins (IgG region), since some CSF patterns showed peaks of basic proteins which are not obvious in the serum of the same patient. About 70 peaks and shoulders of CSF proteins could be detected by the established CIEF technique. The results of CIEF analysis of CSF samples suggested that the technique will be useful as a survey method to detect specific proteins in CSF, which might relate to disorders in the central nervous system.     

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.     

Sample displacement chromatography of monoclonal antibody charge variants and aggregates

The rise of biosimilar monoclonal antibodies has renewed the interest in monoclonal antibody (mAb) charge variants composition and separation. The sample displacement chromatography (SDC) has the potential to overcome the low separation efficiency and productivity associated with bind-elute separation of mAb charge variants. SDC in combination with weak cation exchanging macroporous monolithic chromatographic column was successfully implemented for a separation of charge variants and aggregates of monoclonal IgG under overloading conditions. The charge variants composition was at-line monitored by a newly developed, simple and fast analytical method, based on weak cation exchange chromatography. It was proven that basic charge variants acted as displacers of IgG molecules with lower pI, when the loading was performed 1 to 1.5 pH unit below the pI of acidic charge variants. The efficiency of the SDC process is flow rate independent due to a convection-based mass transfer on the macroporous monolith. The productivity of the process at optimal conditions is 35 mg of purified IgG fraction per milliliters of monolithic support with 75–80% recovery. As such, an SDC approach surpasses the standard bind-elute separation in the productivity for a factor of 3, when performed on the same column. The applicability of the SDC approach was confirmed for porous particle-based column as well, but with 1.5 lower productivity compared to the monoliths.     

A novel approach enables imaged capillary isoelectric focusing analysis of PEGylated proteins

PEGylation has been used as a strategy to enhance pharmacokinetic properties of therapeutic proteins by pharmaceutical industry. Imaged CIEF (iCIEF) is the current industry standard technology for pI determination and charge variant quantification of proteins and antibodies. However, the charge variants of PEGylated proteins merge into one broad peak during iCIEF, most likely due to masking of proteins by the surrounding PEG chain as well as the increased hydrodynamic volume due to PEGylation. Here, we report our novel matrix formula with a combination of glycine and taurine that significantly improved the separation of charge variants in PEGylated proteins. As a result, it is no longer necessary to conduct IEF of proteins prior to PEGylation, which does not reflect the changes caused by PEGylation and purification processes. The novel matrix (glycine and taurine) enables iCIEF analysis of PEGylated proteins in their real conjugated states.     

High‐resolution separation of monoclonal antibodies mixtures and their charge variants by an alternative and generic CZE method

The determination of mAb critical quality attributes (CQA) is crucial for their successful application in health diseases. A generic CZE method was developed for the high‐resolution separation of various mAb charge variants, which are often recognized as important CQA. A dynamic coating of the capillary was obtained with polyethylene oxide (PEO), whereas Bis‐Tris allowed the analysis of mAbs under native conditions at pH 7.0. The effect of PEO and Bis‐Tris concentrations, as well as the nature of the acidic counter ion on the method performance was systematically studied. The %RSD on migration times was below 5% on three different CE instruments using the optimized method. Additional charge variants (in particular acidic variants) were resolved for 10 out of 17 mAbs compared to a reference CZE approach involving the use of ε‐amino‐caproic acid (EACA), triethylenetetramine (TETA), and hydroxypropylmethyl cellulose (HPMC). The amount of basic and acidic charge variants of 17 Food and Drug Administration (FDA) approved mAbs covering a wide range of physico‐chemical properties, e.g., pI between 8.0 and 9.4 and different hydrophobicity, were mainly comprised between 5–15% and 15–30%, respectively. It is noteworthy that applications for the quality control in hospitals as well as for the combination of the immune checkpoint inhibitors nivolumab and ipilimumab were presented.     

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.     

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.     

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.     

A CIEF‐LIF method for simultaneous analysis of multiple protein kinases and screening of inhibitors

Here, a CIEF‐LIF method for multiple protein kinase simultaneous analysis and inhibitors throughput screening with fast rate and low cost is presented. Comparing with CZE, CIEF‐LIF exhibited great focusing ability and high separation efficiency for substrate and phosphorylated peptides, and is applicable for multiple kinases simultaneous analysis regardless of their substrate peptides compositions and charge statuses. Thus, highly sensitive analysis for cyclic adenosine 3’, 5’‐monophosphate‐dependent protein kinase (PKA) and cyclin‐dependent kinase 1 (CDK1) was achieved in CIEF‐LIF analysis with detection sensitivity up to 1.25 mU/μL and 0.4 mU/μL, respectively, two magnitudes higher than that of CZE and comparable with that in nanomaterials or green fluorescent protein‐based kinase assay. Moreover, the inhibition effect of inhibitors on multiple kinases could be simultaneously readout in a single electrophoretic run, with half maximal inhibitory concentration of H‐89 for PKA and Ro‐3306 for CDK1 calculated as 37.0 and 35.9 nM, respectively, consistent with literatures reported. The CIEF‐LIF also exhibited strong anti‐interference ability in human breast cancer cell lysates analysis and simulators such as forskolin and 3‐isobutyl‐1‐methylxantine assessment. Therefore, CIEF‐LIF is desirable for future biological application and clinical diagnostics and drug discovery.     

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.     

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.     

Use of high-performance capillary electrophoresis to monitor charge heterogeneity in recombinant-DNA derived proteins

The separation of charge variants of recombinant DNA-derived (rDNA) proteins by high-performance capillary electrophoresis (HPCE) has been explored with the following examples: human growth hormone (rhGH), a soluble form of a T4 receptor protein (rCD4) and tissue plasminogen activator (rt-PA). The separation of rhGH and deamidated variants were examined over the range of low pH (less than 2.5) to high pH (8.0) on a coated silica capillary. No resolution was observed at pH 2.5 while at pH values of 6.5 or greater the deamidated species were separated. At pH 3.5 the variant was partially resolved but no detector signal was observed at pH 4.5 and 5.0. HPCE was also used to monitor a glycoprotein (rt-PA) with charge heterogeneity presumably due to variable sialic acid content. At a pH value of 4.5, the charge heterogeneity was only observed as peak broadening. For rCD4 multiple peaks were observed at pH 5.5 but no signal was observed at pH 6.5 or above (the pI of rCD4 is 8). These results suggest that HPCE will prove to be a valuable technique for the analysis of charged variants present in rDNA products either as a consequence of natural microheterogeneity or due to degradative processes such as deamidation.