MULTI-PRODUCT OR HIGH THROUGHPUT MAB CHARACTERIZATION METHODS
Electrophoresis
As mentioned previously, heterogeneity of MAbs is monitored as part of the ongoing control system that ensures product quality and manufacturing consistency throughout the commercial life of the product.15,16 Capillary electrophoretic separations, such as capillary electrophoresis with SDS (CE-SDS) and capillary iso-electric focusing (CIEF), have been shown to have significant potential for multi-product usage, negating the need for method development for each product.17,18 CE-SDS analysis can serve as a replacement for silver-stained SDS-PAGE to detect low-level impurities, MAb size variants, and to determine lot-to-lot consistency.19 The high separation power of CE-SDS has been demonstrated by showing baseline resolution between the heavy chain and nonglycosylated heavy chain species present at a low level (2%).20 Recently, quantitative CE-SDS was validated for MAb quality control and stability monitoring.21 CE-SDS also can be performed on microfluidic chip systems for MAb analysis.22 Microfluidic systems offer advantages compared to conventional CE systems, including increased speed of separation, reduced band broadening effects, and low sample consumption.23 Chip-based CE systems can be used for high-throughput analysis because several chips can be applied in parallel.
CIEF is an orthogonal tool used for assessing the charge heterogeneity of MAbs. CIEF usually is performed on commercial capillary electrophoresis instruments, which have a 20–60 cm long capillary and on-column ultraviolet (UV) absorbance detector. Imaged CIEF, or iCIEF, which uses whole-column imaging detection through a charged coupled device (CCD) camera, recently has been commercialized by Convergent Biosciences.24 Although iCIEF is a relatively new technique, it has the potential to assess the overall charge distribution of a variety of MAbs. However, iCIEF methods require specific equipment, and fraction collection for further characterization is indirect. Samples for iCIEF also must undergo additional sample preparation steps, frequently including a buffer exchange before analysis.
Ion Exchange Chromatography
Ion-exchange chromatography (IEC) is widely used for profiling the charge heterogeneity of proteins, yet despite good resolving power and robustness, ionic strength-based ion exchange separations are product-specific and time-consuming to develop. Some improvements have been made to decrease the development time of IEC, such as the development of a quaternary solvent system to simultaneously adjust the pH and salt concentration to reduce buffer preparations during development. In a quaternary solvent system, different combinations of the salt gradient and pH are achieved automatically by programming the HPLC pumps to deliver solvents from four different solvent channels. Figure 2 shows chromatograms obtained on a quaternary solvent IEC system for MAb charge heterogeneity analysis. Pumps A and B were used to adjust the salt gradient, and pumps C and D were used to maintain a target pH. By using the quaternary solvent system, method development time was greatly reduced, from a typical development time of weeks down to four days. Other advantages of developing a method using a quaternary solvent system includes ease of mobile phase composition control, which can greatly reduce the labor needed to make and change a variety of buffers, and the capability of automation for buffer screening during method development. Despite these advantages, the final developed IEC method is product-specific and carries the same disadvantages of conventional IEC, such as limitations in analyzing in-process samples caused by intolerance of the method to various sample matrices and low tolerance to buffer pH changes.16
We have previously reported a novel pH-based separation of proteins by cation exchange chromatography (pH-IEC) that was multi-product, high-resolution, and robust against variations in sample matrix salt concentration and pH.16 Simple mixtures of defined buffer components were used to generate the pH gradients that separate closely related antibody species. This method separated MAb species with a wide range of isoelectric points through a complex retention mechanism, combining both ionic strength and pH. Unlike typical ionic strength elution methods, the pH gradient method was much more generic and could easily separate different components of a range of antibodies using a single method (Figure 3). The multi-product aspect of this method translates to less method development time for new IgG molecules. In addition, the ability of the method to assess charge-heterogeneity at a wide range of sample matrix salt concentrations and pH indicate the suitability of the method for use in evaluating in-process samples. Figure 4 shows chromatograms of samples collected from in-process pools and analyzed using the pH-IEC method. The data obtained from in-process pools using the pH-IEC method are reproducible at various salt concentrations, which is not the case for in-process pools analyzed by conventional IEC and icIEF. Table 2 compares the advantages and disadvantages of conventional IEC, iCIEF, and pH-IEC for charge heterogeneity analysis of MAbs.
Recent work demonstrates the excellent robustness of pH-gradient IEC.25,26 Robustness studies recently have been performed using two column manufacturers, three HPLC instruments, and two analysts over 13 days of analysis. The method performs very well despite changes to column temperature, protein load, and buffer pH. The data from these robustness experiments reflect the expected range for the system suitability results, which reflects the precision of the method that compares favorably to other charge heterogeneity methods (Table 3). The ability of the method to perform well using different column and instrument manufacturers poses a considerable business advantage compared to methods requiring specialized instrumentation and consumables.
Size Exclusion Chromatography
There are several ways to increase the throughput of an analytical method, including reducing method run time. We recently reported a practical SEC method for injecting samples onto the column in rapid succession and gating the detection window to monitor the elution of each sample individually.27 Generally, size-exclusion separations occur in less than a single column volume. Thus, it is possible to minimize the lag time by injecting samples before the previous sample has eluted to increase the throughput, so that at any given instant approximately two samples are eluting through the column. By coordinating the injection and detection time windows, the samples can be kept discrete and significant throughput enhancements achieved, up to nearly two-fold, with a typical run reduced from 30 to 14 min. This approach can be used to increase the throughput for any size exclusion column.
Ultra-high pressure liquid chromatography (UHPLC) has recently been introduced as a liquid chromatography tool to improve separation resolution and reduce overall analysis time. UHPLC uses much higher pressures than conventional HPLC, and the required solvent usage and analyst time is has been reduced because of lessened run times. Until recently, UHPLC was limited to the application of small molecules, but new developments have yielded promising applications for large molecules such as antibodies. Figure 5 shows a chromatogram from size exclusion analysis using a Waters Acquity UHPLC BEH SEC column, in which the run time for analysis of a MAb was only 3 min, compared to typical SEC run times of 15 min or more.
Two-dimensional separations systems involving SEC have been developed to analyze proteins. Recently, automated 2D Protein A–SEC methods for the purification and analysis of MAbs have been developed.28,29 In these methods, multiple protein separation steps were performed automatically. Samples containing antibodies in cell culture fluid were injected onto a Protein A column for antibody recovery, then the purified antibody sample was automatically injected onto the size exclusion column. These 2D methods assessed both titer and size heterogeneity of the MAbs, demonstrating a streamlined workflow, and can be used for a variety of applications, including molecule assessment and clone selection.