Characterization and Comparability Laboratory
University College Dublin
In just three decades, monoclonal antibodies (mAbs) have rapidly transformed the face of modern medicine, growing steadily in use to become the dominant biopharmaceutical product class. More than 70 mAb based products have been approved for use in US and European markets, for the treatment of over 30 targets and diseases, including autoimmune disorders, cardiovascular indications, infectious diseases and cancer.1–3 This growth shows no signs of stopping, with the market for mAbs increasing at a rate of around 10% annually over the past decade and set to be worth an estimated $250 million by 2020.3
Extensive research interest in mAbs has been fueled in large part by their favorable therapeutic characteristics. With enhanced target specificity relative to traditional small molecule drugs, mAbs are highly efficacious yet have a lower potential for off-target effects, making them an attractive option for pharmaceutical development.4 Moreover, improvements in mAb production technologies have significantly improved process yields and reduced manufacturing costs.
As with most large molecule drugs, mAbs are structurally complex and have a sophisticated development and production lifecycle. To protect patient safety, this complexity necessitates additional quality control measures that exceed those required for small molecule drugs. Testing mAbs for the presence of impurities or post-translational modifications involves a variety of characterization techniques geared towards specific types of product.
The need to accommodate comprehensive mAb characterization workflows presents a significant challenge for biotherapeutics analysis laboratories, squeezed by the rapid growth of these complex products. To drive throughput and accelerate time to results, it is essential that these workflows are capable of processing large numbers of samples, while making efficient use of resources. And with more characterization data to gather for mAbs than for small molecule therapeutics, laboratories aren’t simply being asked to do more in the same timeframe, they’re under pressure to know more too. As a result, the most prepared laboratories are embracing more flexible and scalable technologies for high-throughput, high-content biotherapeutic analysis.
Modern UHPLC Workflows for Biotherapeutics Analysis
Ultra High Performance Liquid Chromatography (UHPLC) has become well established as a powerful tool for mAb bioanalysis workflows. Thanks to its exceptional separation resolution, sensitivity and speed, the technique offers greater performance over traditional liquid chromatography techniques. UHPLC is commonly used in combination with a range of different detection technologies, including mass spectrometry (MS), optical detection and charged aerosol detection, and advances in these technologies now allow highly accurate and precise information to be obtained.
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Despite the exceptional analytical performance offered by modern UHPLC systems, throughput remains a significant challenge for many mAb characterization workflows as standard instrument set-ups only allow the use of one stationary phase at a time. Given the need to monitor multiple characteristics for every mAb, this limitation means that separate analytical runs must be performed sequentially, adding analytical switching steps and extending the total analysis time (Figure 1). Greater throughput could, in theory, be achieved using multiple instruments. However, for many organizations, limited budgets – and even limited laboratory space – mean that investing in additional equipment simply isn’t an option.
Instrument vendors have recently developed solutions to tackle this workflow bottleneck. The latest generation of UHPLC systems incorporate a second channel for separate simultaneous sample analysis using the same instrument. By incorporating two pumps and detector systems, these systems permit analysis using two separate liquid chromatography (LC) methods through two flow paths, allowing separate experiments to be performed in parallel (Figure 1). In this way, dual LC systems are expanding the scope of UHPLC characterization methods and improving lab productivity, sample information, efficiency and cost per sample.
Using Dual Channel UHPLC for Simultaneous Orthogonal mAb Characterization
The efficiency savings that can be gained using dual channel UHPLC workflows were recently demonstrated in a characterization study of two mAbs, bevacizumab and infliximab, using the Thermo Scientific Vanquish Flex Duo UHPLC system for dual LC. Each mAb was simultaneously characterized by two orthogonal methods, employing different elution solvents and different instrument parameters (including different data collection wavelengths, gradients, isocratic conditions and total run times). A combination of size exclusion chromatography (SEC), strong cation exchange (SCX) chromatography and reversed phase (RP) chromatography, coupled with UV detection, was employed.
SEC is commonly used to quantify protein aggregation to determine the extent of protein denaturation that may have occurred during manufacturing or storage. These structural changes can jeopardize product safety and efficacy, and their identification is essential. SCX chromatography is typically used for charge variant analysis, and works by separating molecules with different charge states that may have resulted from sequence truncations or glycan structure modifications. Lastly, RP chromatography is often used to analyze intact mAbs for stability studies and structure confirmation.
Bevacizumab was evaluated following two analysis methods: SEC aggregate analysis on one channel and charge variant analysis on the second channel. A second mAb drug, infliximab, was also evaluated using SCX charge variant analysis on one channel and RP intact analysis on the second channel. Both sets of experiments were performed with ten technical replicates each.
Sample Preparation
Bevacizumab (25 mg/mL) was diluted 1:10 in water. Infliximab was prepared at a concentration of 10 mg/mL in water. Diluted mAb samples were aliquoted and stored at -20 °C.
Size Exclusion Chromatography Conditions
Mobile phase (isocratic): 100 mM sodium phosphate, pH 6.8 in 300 mM NaCl; flow rate: 0.25 mL/min; run time: 16 min; column temperature: 30 °C; autosampler temperature: 5°C; UV wavelength: 214 nm and 280 nm; injection volume: 2 μL of 25 mg/mL bevacizumab; injection wash solvent: methanol/water (20:80 v/v).
Strong Cation Exchange Chromatography Conditions
Gradient methods were employed for infliximab and bevacizumab. Mobile phase A: 10-fold dilution of CX-1 buffer A (pH 5.6) with deionized water; mobile phase B: 10-fold dilution of CX-1 buffer B (pH 10.2) with deionized water; run time: 30 min; flow rate: 0.2 mL/min (infliximab), 0.3 column mL/min (bevacizumab); column temperature: 30 °C; autosampler temperature: 5 °C; UV wavelength: 280 nm; injection volume: 1 μL of 10 mg/mL infliximab, 2 μL of 25 mg/mL bevacizumab; injection wash solvent: methanol/water (20:80 v/v).
Reversed Phase Chromatography Conditions
A gradient method was employed. Mobile phase A: water/TFA (99.9:0.1 v/v); mobile phase B: acetonitrile/water/TFA (90:9.9:0.1 v/v); run time: 10 min; flow rate: 0.6 mL/min; column temperature: 80 °C; post-column cooling temperature: 40 °C; autosampler temperature: 5 °C; UV wavelength: 214 and 280 nm; injection volume 0.5 μL of 10 mg/mL infliximab; injection wash solvent: methanol/water (20:80 v/v).
Analysis
Chromatograms obtained for the dual LC-UV analysis of bevacizumab and infliximab are shown in Figures 2 and 3, respectively. Analysis of the main peak characteristics obtained using both channels for the two simultaneous analyses are also presented.
The low percentage relative standard deviation (RSD) values for the measured parameters for both sets of data highlight the excellent reproducibility across replicates and the robust performance of each chromatographic channel, even during simultaneous analyses. The chromatograms displayed precise and specific peaks for each method, demonstrating the suitability of this method for obtaining orthogonal characterization data for the two mAbs studied.
The ability to simultaneously analyze samples using two different methods when characterizing mAbs offers time and cost savings as well as high-throughput possibilities without changing validated approaches.
Furthermore, sample preparation can be reduced by using the same set of samples in different analyses at one time. In quality control environments where confidence in results is just as important as rapid turnaround times, generating high-throughput data without compromising on analytical performance is highly beneficial.
Using Dual Channel UHPLC for Tandem LC-MS Workflows
Peptide mapping is another important mAb characterization technique that provides primary sequence confirmation and enables the identification and quantitation of post‑translational modifications such as oxidation, deamidation and glycosylation. Despite the impressive characterization data that can be obtained by UHPLC peptide mapping workflows, these methods typically require extended column reconditioning steps, adding time to overall workflows.
Column reconditioning steps or washing procedures are an essential part of many LC sequences that employ gradient elution. These steps are necessary to re-equilibrate the column for the next sample, while reducing the potential for residues to remain on the column and helping to maintain stable backpressures and deliver consistent separations. It is recommended that reconditioning steps use at least five column volumes of eluent to sufficiently equilibrate the stationary phase (5), although if a mobile phase is buffered or contains an ion pair reagent, the minimum equilibration time may be longer.
While individual reconditioning runs may take several minutes, over the course of an entire analytical sequence these steps can add hours to workflows, without resulting in the collection of meaningful data.
With many laboratories making significant investment in powerful MS detection technologies, the fact that these technologies remain idle during reconditioning steps is a significant drawback of single channel LC workflows.
The option to use two separate LC channels, controlled by a switching valve, allows analysts to do more with less by running the same validated method across two pumps. Because both channels are used for the same method, one can be used for the collection of data, while the second can simultaneously be used to prepare the column for the subsequent analytical run (Figure 4). This can even allow for additional column washing steps, helping to improve the consistency of subsequent measurements. This allows analysts to take full advantage of MS technologies to deliver shorter turnaround times and lower cost per sample.
This potential to accelerate LC-MS workflows was recently demonstrated in a peptide mapping study of infliximab, using the Thermo Scientific Vanquish Horizon Duo UHPLC system for tandem LC-MS.
Sample Preparation
A 50 μL infliximab sample, adjusted to 2 mg/mL with water, was diluted 1:4 with Thermo Scientific SMART Digest buffer. The solution was transferred to a reaction tube containing 15 μL of the SMART Digest resin slurry, corresponding to 14 μg of heat-stable immobilized trypsin. Tryptic digestion was performed at 70°C for 45 min at 1400 rpm. Following digestion, the reaction tube was centrifuged at 7000 rpm for 2 min, the supernatant was transferred to a new tube, and the centrifugation step was repeated. The non-reduced sample was diluted with 0.1% formic acid (FA) in water to a final protein concentration of 100 ng/μL, and 1.0 μg was loaded on the column for all runs.
LC Conditions for the Separation of the mAb Digest
Gradient methods were employed for the analytical and reconditioning sequences. Mobile phase A: Water + 0.1% formic acid; mobile phase B: water/acetonitrile (10:90 v/v) +0.1% formic acid; flow rate: 0.4 mL/min; column temperature: 60°C; UV wavelength: 214 nm.
Analysis
Five total ion current (TIC) chromatogram replicates obtained on two columns with automated alternating column reconditioning are shown in Figure 5 for the separation of tryptic digested infliximab.
The tandem LC-MS peptide mapping workflow enabled an increase in throughput of up to 40% without changing the gradient of the existing method. This highlights the significant time savings that can be made using dual channel UHPLC set-ups.
The RSD values for the retention time data obtained in tandem column operation were found to be below 0.11%. This compares to values of 0.045% and 0.039% for the single column set-up. The highest RSD values were obtained for polar tryptic peptides eluting between 0 and 14 min (up to 0.18%), while the lowest RSD value was obtained for the heavy chain peptide eluting at 30.85 min (0.064%). The average absolute retention time shift between the two columns was 0.023 min, demonstrating the excellent measurement consistency of the tandem LC-MS set-up. An average peak area RSD value of 2.47% demonstrates the suitability of the approach for quantitative data analysis.
Conclusion
Combining orthogonal chromatographic methods using dual channel UHPLC systems offers distinct advantages for in-depth biotherapeutics characterization, saving time and helping quality control workflows operate more efficiently.
Likewise, when used for tandem LC-MS applications such as peptide mapping, these systems are driving productivity while maintaining exceptional accuracy, precision and reproducibility.
Whether they are used for orthogonal or tandem analysis, dual channel UHPLC systems are rapidly generating highly consistent data to support fast and effective decision-making.
Author Biographies
Dr. Amy Farrell is a biopharmaceuticals development application team lead in NIBRT Ltd., Ireland. Amy received her PhD in Bioanalytical Science from Dublin City University in 2016 based on her research activities using quantitative LC-MS techniques to facilitate enhanced bioprocessing of therapeutic proteins. Amy has over a decade of experience working in both the pharmaceutical and biopharmaceutical industries.
Dr. Sara Carillo is the applications development team leader in the Characterization and Comparability Laboratory in NIBRT, Ireland. Sara completed her PhD in Chemical Science at the University of Naples in 2013 working on bacterial glyco-conjugates structural characterization. In 2015, Sara joined Dr. Jonathan Bones’s research group in NIBRT, working on CHO cell glycome and biotherapeutics characterization.
Dr. Jonathan Bones received his PhD in Analytical Chemistry from Dublin City University in 2007. Jonathan then moved to NIBRT, working under the mentorship of Prof. Rudd. In 2010, Jonathan was appointed the John Hatsopoulos Research Scholar within the Barnett Institute, Boston, working under the mentorship of Prof. Karger. Jonathan returned to NIBRT in 2012 and is the PI of the Characterization and Comparability Laboratory and an Associate Professor in the School of Chemical and Bioprocess Engineering at University College Dublin.
References
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