Monoclonal antibodies (mAbs) are currently under development by a number of pharmaceutical and biotechnological companies and have proven a successful class of therapeutic agent - owing to their high specificity and low side effects - when compared to small molecule drugs. While the therapeutic power of mAbs is high, production comes with a level of product variation and related impurities. Such impurities include lysine truncation fragmentation, and glycation. These processes can negatively affect the integrity of mAbs being produced, and the heterogeneity of purified antibodies based upon parameters such as chemical modifications of selected amino acid sites is of great interest to the biotechnology industry as it provides information on product quality. Enzymatic modifications such as deamination result in a change in the net charge on mAbs, which leads to the formation of charge variants during manufacture, which is threatening to stability. Understanding antibody charge variants has gained considerable attention due to the potential influence they have on the structure, stability, binding affinity, and reactivity of mAb. Developing methods for the stratification and separation of charge variants will aid with establishing manufacturing consistency between lots, and will help comparable studies follow process alterations.
Conventional methodologies for the separation of mAb charge variants such as salt gradient cation exchange chromatography, have proven effective to a degree, but such methods have to be tailored for individual mAbs. This is not suited to a fast-paced biotechnology drug-development program, leaving much to be desired for efficiency in mAbs characterization. Following the discovery of a pH-gradient based method for variant separation, which showed marked improvements, a number of technologies have been developed that further improve the efficiency of this method. The development of pre-formulated buffers for pH gradient – based separation has worked to simplify the development of ion exchange chromatography (IEX) for the separation of mAbs based on their charge.
Three key areas are crucial in the development of such a pH gradient buffer. The first is the pH range covered by the buffer. Modern ranges developed can be as wide as 5.6 to 10.2. This range enables the characterization of mAbs with a far wider range of isoelectric points – making it more suitable for laboratories that expect a high output of mAb’s with a wide range of isoelectric points. The second feature is the linearity in solvent gradient programmed in the pump being reflected in the column. This is not a trivial feature, as personnel using IEX know how difficult this is to achieve manually. This genuine linear pH gradient allows for simple fine-tuning of the pH range around the mAb and variants being worked with at the time, which enables the adjustment of gradient time according to the method resolution requirements. The third feature lies in the mobile phase preparation in that modern pre-formulated pH buffers only need to be diluted by a factor of 10 before the mobile phase is ready to use. Innovations like these work to position pH-gradient IEX ahead of salt-gradient methods in terms of workflow efficiency and the ability to conduct more generic screening easily.
This article outlines methods to push the throughput of pH gradient IEX even further. To achieve this, a small-particle-size column was operated on an ultra-performance liquid chromatography (UPLC) system. The system was fully biocompatible - suitable for the analysis of intact proteins. The combination of low gradient delay volume and high precision gradient formation made it an ideal system for high throughput analysis with gradient elution. High resolution pH gradient separations are obtained with 30 minute gradients and relatively long columns. Fast separation of mAb variants were demonstrated with no significant loss in resolution. In this case, the pH gradient from 5.6 to 10.2 was completed in under 2 minutes, and the resolution between variants was still satisfactory, despite the short analysis time.
Materials and Methods
Efforts were made to demonstrate methods that could streamline the analysis and implementation time for UHPLC separation of mAb by charge. Traditional salt gradient ion exchange methods for charged variant analysis usually have run times of one hour or more. Each individual mAb sample will have a method that is optimized for the highest resolution of the charged variants in that particular mAb. The method development process would involve screening for the optimum pH for separation, then further development of the gradient. This process is very time consuming and could take several weeks to optimize just one method. The pH gradient optimization involves one generic wide pH range screening run of 10 minutes followed by a faster optimization run based on the elution pH from the screening run.
The UHPLC system used consisted of; a Vanquish system base, binary pump H with default mixer, split sampler HT, column compartment H, and diode array detector HL. Table 1 below outlines the chromatographic and detector conditions, as well as the data processing software used for UHPLC. The mAbs used were; bevacizumab (25 mg/ml), cetuximab (5 mg/ml), infliximab (10 mg/ml), transtuzumab, (21 mg/ml), and mAb A (21 mg/ml).
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Chromatographic Conditions |
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Column: MAbPac SCX-10 RS, 5 μm, 2.1 × 50 mm (P/N 082675) |
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Buffers: CX-1 pH-Gradient buffer A (pH 5.6) 125 mL (P/N 083273) CX-1 pH-Gradient buffer B (pH 10.2) 125 mL (P/N 083275) |
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Mobile Phase A: CX-1 pH-Gradient buffer A (pH 5.6) diluted 10x in deionized water |
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Mobile Phase B: CX-1 pH-Gradient buffer B (pH 10.2) diluted 10x in deionized water Column Compartment Temperature 30 °C, forced air |
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Detector and Conditions |
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Detector: 10 mm Standard Flowcell (P/N 6083.0100) |
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Detection Wavelength: 280 nm |
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Data Acquisition Range: 5 Hz (for flow rate ≤ 0.5 mL/min) and 50 Hz (for flow rate ≥ 1.0 mL/min) |
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Response Time: 2 s (for flow rates ≤ 0.5 mL/min) and 0.1 s (for flow rates ≥ 1.0 mL/min) |
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Data Processing |
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Software: Dionex Chromeleon 7.2 Chromatography Data System (CDS) |
Table 1: Chromatographic conditions, detector conditions, and data processing for UHPLC.
Results and Discussion
The development of advanced buffers for pH-gradient based IEX allows the simplification in method optimization and development. The optimized buffer components result in a linear pH gradient in the column. This is advantageous over nonlinear in-house buffers produced in the laboratory, which make method optimization difficult because the user cannot be certain of the effects of any changes in the programmed gradient. When this is combined with a high quality, low dead volume UHPLC pumping system, methods can be developed quickly with final run ties as low as 2 minutes. It is recommended to perform a general screening from pH 5.6 to 10.2 when the pH at which a particular mAb elutes is not known. Here, the generic screening was run in 10 minutes at 0.45 mL/min. Satisfactory separation of the charge variants was achieved during the first run. As shown in Figure 1, for cetuximab and infliximab several charge variants could be resolved with sufficient resolution.
Following the generic screening, efforts were made to decrease the analysis cycle time and improve resolution. To achieve this, the pH range and the gradient slope were modified. A narrower pH window allowed for a reduction in run time, whereas a shallower gradient slope provided better resolution. The gradient slope of the generic screening between pH 5.6 and 10.22 was 22.2 %B/mL (where %B is the amount of B eluent), whereas the improved analysis was obtained with gradient slopes of 8 and 10(%B)/mL. Figures 2a, 3a, 4a and 5a, show the results obtained by this approach. The number of resolved variants was larger in the initial screening. Steps were then taken in order to create fast analysis cycles compatible with high throughput, with a view to create a 2.5-minute gradient time or low, and a total analysis time of no more than 4 minutes. The results for these runs are shown in Figures 2b, 3b, 4b and 5b.
The purpose was the development of a method suitable for high-throughput that can run at least 300 samples a day. For this reason, a high flow rate was implemented which allowed for the running of short gradients with relatively shallow gradient slopes required to preserve selectivity between charge variants. In addition to this, column equilibration – which is directly dependent on the column volume of mobile phase following through the column – which is reached quicker at higher flow rates on shorter columns.
The chromatographic pattern between methods at moderate and high flow rate was preserved and the separation capabilities of different methods were compared based on the resolution between charge variants. Since in several instances peak pairs were overlapping, and it was not always possible to measure peak width at half height or at the baseline, here resolution used was based on statistical moments.
With trastuzumab, ultra-fast separation was accompanied with resolution loss of approximately three percent. This was higher in the case of complex variants as observed with cetuximab (approximately 13 percent). Infliximab resolution decreased by approximately 11 percent, however separation of the 5 main charge variants and 2 minor ones was achieved in 1 minute. This was achieved via the running of the column at 1.2 mL/min with 0.8 min gradient time. In the case of bevacizumab, the ultra-fast separation approach even yielded to ~ 4 percent improved resolution. The better resolving power can be explained by a slightly narrower pH range and a shallower gradient slope. mAb A was analyzed only with the generic screening and high flow rate (1 mL/min) method. The high-throughput method provided 19 percent better resolution on average of 5 charge variants.
Conclusion
Charge variant analysis has been improved with the use of a modern commercially available pH buffer system. Conventional salt gradients can take weeks to develop a method for each individual mAb with traditionally long run times. With this technique, fast methods can be generated in a few minutes on a wide range of mAb’s. The development of novel and improved techniques for the efficient separation of mAb variants based on charge will help in the production of high quality mAbs for use in therapeutics. The ultra-fast variant separations described are achieved because of several advances in chromatography methodologies and technology. The mechanism of pH gradient chromatography lends itself to the use of shorter, faster columns. The availability of high pressure rated small particle size ion exchange columns are a perfect match to pH gradient methodology. The commercial buffer formulations used here form a linear gradient which allows intelligent optimization of the methods
Dr. Ken Cook received his Ph.D. in Biochemistry from Newcastle University, where he became a lecturer in protein chemistry. Currently, Ken is the European Bio-Separations expert for Thermo Fisher Scientific where biopharmaceutical applications to characterize protein and nucleotide based bio-therapeutics are primary application fields.
Dr. Frank Steiner received his Ph.D. in Chemistry in 1995 before becoming a postdoctoral research fellow at the CEA, Saclay in France where he focused on elementary and isotopic analysis by IC and IC-ICP/MS. At Thermo Fisher Scientific, Frank is Scientific Advisor for HPLC, coordinating scientific collaborations with external partners to advance UHPLC technologies and applications.
