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These new analytical methods can reduce analysis and product development time.
Monoclonal antibodies represent a significant portion of sales in the biopharmaceuticals market. Ever-growing biotechnology pipelines have greatly increased demand for higher throughput, multi-product protein characterization methods. By taking a holistic approach to improving the analytical process, one can identify strategies to increase throughput and reduce development times and the costs of analytical methods. This article discusses recent advances in high-throughput and multi-product liquid chromatography and electrophoretic methods for protein characterization. In addition to advances in analytical methodology, technological advancements in sample preparation throughput and data handling automation add value and time savings to the overall characterization process, which will enable increased productivity and faster product development.
Monoclonal antibodies (MAbs) are an important class of therapeutic proteins in biotechnology and have been developed to treat a variety of indications to fill significant unmet medical needs.1 MAbs generally are target-specific and well tolerated with a relatively long half-life, contributing to their success in drug development. Of the classes of immunoglobulins, IgG1 is the most commonly used for pharmaceutical and biomedical purposes.2
In recent years, increased understanding of disease and advances in drug discovery have resulted in continuously growing pipelines for potential therapeutic antibody products. To keep up with the increasing demand for protein characterization, high-throughput and multi-product characterization methods are being developed. If used correctly, these methods can reduce analysis and development time and thus accelerate the progress of therapeutic proteins toward the clinic.
Several guidance documents have been issued by regulatory agencies recommending approaches for protein characterization.3,4 Although guidance documents are valuable to the industry, other publications have emerged that more clearly identify methods that are useful for characterizing MAbs.5 These orthogonal assays include potency, identity, and impurity assays that evaluate critical quality attributes (CQAs) such as size and charge heterogeneity.5 These CQAs are part of the overall target product profile, which is based on the desired clinical performance. Appropriate control of CQAs is a common review concern for both investigational new drug (IND) and license applications.6,7 Product quality characteristics encompass a wide variety of product variants that include product-related species and product-related impurities.8 For example, aggregation is a carefully monitored product variant from the earliest stages of clinical development because of the possibility of eliciting an immunogenic response in the patient.
Many of the recommended protein characterization assays are based on liquid chromatography methods such as ion exchange chromatography (IEC) for charge heterogeneity analysis, size exclusion chromatography (SEC) for size heterogeneity, and reverse-phase high performance liquid chromatography (RP-HPLC) for peptide mapping.9 This article primarily will focus on multi-product and high-throughput charge-based methods for analyzing charge heterogeneity and size exclusion methods for quantifying size heterogeneity, in particular aggregates or high molecular weight species (HMWS). These methods are among the most frequently used tests for lot release of drug substance and drug product,5 as well as during formulation and process development. Several multi-product analytical methods for MAb size and charge assessment are shown in Table 1.
Table 1. Multi-product analytical methods for monoclonal antibody size and charge assessment
Before an analytical method can be incorporated into a characterization platform or a quality control system, it must first demonstrate that the method is suitable for its intended purpose. Guidelines for validating analytical methods have been published in the US Pharmacopeia,10 by the US Food and Drug Administration,11,12 and in published reviews.13 The guidelines published by the International Conference on Harmonization (ICH) have established a uniform understanding of the performance characteristics that are evaluated in the course of validation.14 Although high-throughput and multi-product methods can save time and add value to a business process, these methods also must be evaluated considering regulatory requirements and validation procedures. In other words, the validatability of these methods must be assessed before implementation.
A generalized sample analysis workflow is shown in Figure 1. Typically, protein samples are submitted, prepared, and analyzed, and then results are reviewed and reported. When developing high throughput or multi-product analytical methods, it is important to keep a holistic viewpoint of the sample analysis workflow to optimize the total process. For example, if a new method is developed with reduced run time but with increased sample preparation steps, then the value of reduced run time could easily be negated.
Figure 1. Schematic of a generalized sample analysis workflow
When exploring high-throughput and multi-product methods, investigation into cost savings is aided by a holistic view of the organization. Methods that use the existing analytical equipment in an organization may be more advantageous to develop than methods that require extensive capital investment on new instruments and consumables. In addition to capital costs, analyst experience should be leveraged when implementing new methods. Many biotechnology and pharmaceutical companies are well equipped with many types of liquid chromatography instruments and analysts that are trained to use them. Thus, the development and implementation of high-throughput and multi-product liquid chromatography methods that leverage existing facilities may result in greater time and cost savings compared to novel methods that do not use existing resources. Over-engineering a solution should be avoided; an existing technology should not be overhauled when a minor update would suffice to meet business needs.
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.
Figure 2. MAb samples analyzed by quaternary solvent IEC. Samples were digested with carboxypeptidase B before analysis. Flow rate was set to 0.5 mL/min. Sample loading was 20 Âµg. Column temperature was 40 Â°C. Solvent A: water. Solvent B: 0.5 M NaCl. Solvent C: 20 mM ACES, 20 mM MES, 20 mM Tris, 20 mM HEPES, pH 5.5. Solvent D: 20 mM ACES, 20 mM MES, 20 mM Tris, 20 mM HEPES, pH 8.5.
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
Figure 3. Elution profiles for five MAbs with different pI values using a single pH gradient ion exchange method. Column: ProPac WCX-10, 4 mm Ã 50 mm, 10 Âµm particle size. Column temperature: 25 Â°C. Flow rate: 1.0 mL/min. Sample load: 20 ug (20 ÂµL injection), with samples diluted to 1 mg/mL with mobile phase A. Gradient: 0â100% B in 16 min. Mobile phase: 9.6 mM Tris, 6.0 mM imidazole, 11.6 mM piperazine A) at pH 6.0, B) at pH 11.0.
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.
Figure 4. MAb samples from in-process anion exchange chromatography pools (Q-pool) analyzed by pH-IEC. Samples were diluted to ~1 mg mL-1, with no adjustment to pH. Protein A purified samples show equivalent profiles compared to samples analyzed directly from in-process pools.
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.
Table 2. Advantages and disadvantages of conventional ion exchange chromatography (IEC), imaged capillary isoelectric focusing (iCIEF), and pH-gradient ion exchange chromatography (pH-IEC).
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.
Table 3. Precision in terms of absolute standard deviation range (Â±3 SD) of main peak relative area for conventional ion exchange chromatography (IEC), imaged capillary isoelectric focusing (iCIEF), and pH-gradient ion exchange chromatography (pH-IEC) for three MAbs.
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.
Figure 5. Size exclusion chromatography (SEC) analysis of a MAb using a Waters Acquity ultra high pressure liquid chromatography instrument. Waters UPLC BEH column dimensions were 4.6 Ã 150 mm, with 1.7 Âµm particles. The column was run at ambient temperature. The mobile phase consisted of 200 mM potassium phosphate, 250 mM potassium chloride, pH 6.2. Flow rate was set to 0.8 mL/min. Sample loading was 10 ug. Relative peak areas for high molecular weight species (HMWS), monomer, and low molecular weight species (LMWS) are tabulated.
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.
In addition to high-throughput and multi-product analytical methods, the use of robotics for sample preparation automation may further reduce sample analysis time and cost. There are several companies that provide liquid handling automation instruments, including LEAP Technologies and TECAN. Figure 6 shows a LEAP Technologies CTC PAL liquid handling system, which is intended for HPLC and GC usage and capable of fraction collection and injection. The CTC PAL instrument is capable of on-the-fly sample preparation, such as protein dilution and digestion. On-the-fly sample preparations are viable if the sample preparation takes less time than the analytical method. For sample preparations that take longer than the analytical run time, batch sample preparation can be performed using robotic liquid handling systems such as the TECAN Freedom EVO, which can handle multi-well plates for increased sample throughput. Robotic liquid samplers can increase reproducibility, efficiency, and safety compared to manual handling of samples.
Figure 6. LEAP Technologies CTC PAL sample preparation robotics system. The system interfaced with a Dionex U3000 HPLC, with both CTC PAL system and HPLC controlled by Dionex Chromeleon chromatography management software. MAb samples were digested and diluted by the CTC PAL and injected directly onto the HPLC for IEC analysis.
The final steps to most characterization workflows include data analysis and report generation. Several software packages are available that are designed to reduce the time necessary to complete post-data acquisition tasks. For liquid chromatography applications, commercially available chromatography data software, such as Dionex's Chromeleon chromatography management software and Waters Corporation's Empower chromatography data software, include features such as automated peak integration and one-click report generation. The Chromeleon software also has an extension pack that includes validation report templates and sequences, enabling increased automation of method validation. More companies are implementing electronic laboratory notebooks, which have advantages over traditional laboratory notebooks such as ease of data sharing and collaboration, streamlined review and witnessing processes, standardized documentation, and long-term data preservation.
MAbs are under development for a wide range of indications, and ever-growing biotechnology pipelines have greatly increased demand for high throughput, multi-product protein characterization methods. By taking a holistic approach to improving the protein characterization process, one can identify strategies to increase value and reduce the development time of an analytical method. Implementing high-throughput and multi-product liquid chromatography methods that use available knowledge and resources typical in a biopharmaceutical facility can address the increasing demand for protein characterization studies, and these methods can be readily validated following regulatory guidelines. Further technological advancements in sample preparation throughput and data handling automation adds value to the overall characterization process, which will enable faster product development and shorter timelines to clinic.
We would like to acknowledge the many people involved in our work, including but not limited to Ed Bouvier at Waters Corporation, Mark van Gils and Guillaume Tremintin at Dionex Corporation, and Peter Smith at LEAP Technologies, for technical input and assistance.
Jennifer C. Rea, PhD, is an associate scientist, G. Tony Moreno is a senior research associate, Yun Lou is a research associate, Rahul Parikh is a research associate, and Dell Farnan, PhD, is a senior scientist and associate director, all at Genentech, Inc., Protein Analytical Chemistry, South San Francisco, CA, 650.225.6378, firstname.lastname@example.org
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