Application Note LCMS-87 Automated Acquisition and Analysis of Data for Monitoring Protein Conjugation by LC-MS Using BioPharma Compass

Transcription

1 Application Note LCMS-87 Automated Acquisition and Analysis of Data for Monitoring Protein Conjugation by LC-MS Using BioPharma Compass Abstract Here we describe the development and use of an automated UPLC-MS-based method for the monitoring and analysis of the conjugation of small molecules and peptides to proteins. The automation is implemented using Biopharma Compass software and allows for data acquisition, processing and analysis to occur with minimal user intervention (1). The software ensures that all acquisition parameters, and any future processing that may occur, are traceably linked to the samples analyzed. Post-acquisition, samples are automatically processed in a number of steps in order to ultimately provide a judgement on the outcome of the experiment according to criteria specified by the user. In the example reported here, albumin was conjugated with a fluorescent dye. The success of this experiment was judged according to the percentage of correct conjugation state that was detected at different time points during a time course experiment, with the correct conjugation state assigned as being a 1:1 ratio of albumin:dye. The intact mass of all protein conjugates produced was calculated and used to determine the conjugation ratios for the various conjugation states that were produced at a given time point, based on signal intensity. From this, the percentage of protein that was detected in the correct conjugation state could be used as an indication of the purity of the final conjugated product. Authors Malcolm J. Saxton, Ryan Hylands, Karl Nichols, Joanne Waters Novozymes Biopharma UK Ltd., Nottingham, UK Stuart Pengelley, Wolfgang Jabs Bruker Daltonik GmbH, Bremen, Germany Keywords BioPharma Automation Quality Control Conjugation Instrumentation and Software BioPharma Compass O-TOF Instruments

2 Once processing is complete the data is presented in a graphical interface, in which colour is additionally used to inform the user of the conjugation state of the sample and to highlight which samples may require further user intervention. The automation of acquisition, data processing and analysis allow for both the simple confirmation of correct sample conjugation and the analysis of complex conjugation time course experiments to be carried out reproducibly with the minimum amount of user intervention, saving significant resources and ensuring high quality data and interpretation. Introduction The beneficial application of therapeutics is often limited by short biological half-life and poor bioavailability of the active ingredient. Rapid drug clearance from the body due to short half-life can result in higher and more frequent dosing, causing peaks and troughs of drug concentration in the patient. Albumin drug fusion or conjugation technology can be employed to extend the circulatory half-life of the target active pharmaceutical ingredient, resulting in less frequent administration and reduced side effects. Due to albumin s naturally long half-life of 19 days, in humans, fusion or conjugation of therapeutic proteins to the albumin molecule has been shown to extend drug half-life. The extended half-life of albumin, like IgG, is due to the large size of the molecule and its ph-dependent interaction with the major histocompatibility complex (MHC) class I-related neonatal receptor (FcRn). Both albumin and IgG are protected from intracellular degradation through binding to FcRn in acidic endosomes (2). Conjugation allows small proteins, peptides and importantly small molecules to be attached to albumin either stably or reversibly to allow release at specific locations within the body. For both product consistency and optimal half-life extension it is important to fully understand the conjugation reaction being used to attach an active ingredient to albumin. Analysis of these albumin drug conjugates is essential to ensure detailed knowledge of the products, and one of the analytical techniques used is UPLC-MS in combination with Biopharma Compass. UPLC-MS provides a quick and robust workflow to follow conjugation reactions and determine the conjugation state (conjugates per albumin molecule) of the final product, while Biopharma Compass allows fast, reliable, and quantitative characterization of biopharmaceuticals in a high throughput, automated manner. In addition, data is captured and stored via fully auditable and traceable processes. Reports can be customized for each type of analysis to highlight abnormal or unexpected results. In the work described here, automated visual reports were generated which allow product purity and identity to be observed at a glance for the entire batch of samples. Experimental Conjugation Fluorescein-5-Maleimide (F5M) was dissolved in dimethylformamide (DMF) before being diluted in phosphate-buffered saline (PBS) to a concentration of 2925 nmol/ml at ph 7.0. The F5M solution was then added to recombinant Albumin (ralb, 1 mg/ml) at ratios of 1:1, 5:1, 10:1 and 20:1. These reactions were incubated at 25 C and a 10 µl sample was automatically taken every 15 mins for UPLC-MS analysis. UPLC-MS Conjugated ralb were analyzed to determine conjugation state by UPLC-MS. LC separation was carried out using a Waters Acquity on a BEH mm ACQUITY BEH 1.7 µm C4 column and a 15 min analytical gradient. Table 1: Experimental conditions for UHPLC. HPLC Column Column Oven 50 C Flow Rate Solvent A Solvent B Run Time Mass Spectrometer Waters Acquity UHPLC BEH 50 x 2.1 mm Acquity BEH 1.7 µm C4 column 0.4 ml/min 0.1% FA in H2O 0.1% FA in ACN 0 min 5% B 0.5 min 5% B 11 min 50% B 11.1 min 80% B 13 min 80% B 13.1 min 5% B 13 min 5% B Bruker microtof II The experimental and data processing workflow is summarized in Figure 1. Eluted proteins were directly introduced to a Bruker MicrOTOF II mass spectrometer via an ESI source. All instrument control and sample tables were controlled using Bruker s BioPharma Compass software with previously optimized settings.

3 Summary of experimental and data processing workflow Data Analysis Conjugation Experiment Recombinant Human Albumin Fluorescein- 5-Maleimide All data was processed in an automated manner in accordance with parameters which were submitted by the user. These sample-specific parameters supplement the pre-set method and must be provided for each sample or batch of samples, usually submitted in the form of an Excel spread sheet as shown in Figure 2. BioPharma Compass Conjugation Optimization LC-MS Waters Acquity Bruker microtof II Data Processing Protein Deconvolution Conjugation State Determination Graphical Display of Data Conjugation State Product Quality In the example shown, the user supplied information regarding the retention time and expected mass range of the peaks of interest which was used to direct the automated deconvolution of the raw data by the Maximum Entropy algorithm. Further information was required regarding molecular weight (Mr of the unmodified protein was needed as a reference for each sample in addition to a window of tolerance) and conjugates expected in the sample (conjugate mass and the maximum number of conjugates to be screened for) in order to judge the success of the experiment. A variety of metrics were calculated to determine the success of the conjugation, including: Maximum number of conjugates per molecule Average number of conjugates per molecule Most abundant number of conjugates Percentage correct conjugation state Percentage intensity due to conjugated ralb Evaluation Effect of conditions on conjugation efficiency Temperature Concentration Figure 1: Summary of the experimental and data processing workflow for studying protein conjugation. Fluorescein-5-Maleimide (F5M) was mixed at four different ratios with recombinant Albumin (ralb) and incubated at 25 C to allow conjugation. 10 µl sample was taken every 15 mins for UPLC-MS analysis under the control of BioPharma Compass, which allows data acquisition, processing and reporting to be performed in an automated manner. Key workflow parameters Timepoint Sample Description Position Injection Volume [ul] Relevant RT Range Start Relevant RT Range End MZ Deconvolution Start MZ Deconvolution End Accepted Mass Deviation in Da Unmodified Protein Mr Maximum Number of Conjugates Conjugate Mass Main Conjugate Number 1 ralb:f5m 1:1 B ralb:f5m 1:5 B ralb:f5m 1:10 B ralb:f5m 1:20 B ralb:f5m 1:1 B ralb:f5m 1:5 B ralb:f5m 1:10 B ralb:f5m 1:20 B ralb:f5m 1:1 B ralb:f5m 1:5 B ralb:f5m 1:10 B ralb:f5m 1:20 B ralb:f5m 1:1 B ralb:f5m 1:5 B ralb:f5m 1:10 B ralb:f5m 1:20 B ralb:f5m 1:1 B ralb:f5m 1:5 B ralb:f5m 1:10 B ralb:f5m 1:20 B Figure 2: Key workflow parameters for the automated acquisition, processing and evaluation of data as submitted to BioPharma Compass via an Excel spreadsheet. Parameters are shown for the first 5 of 10 timepoints.

4 Results Processed data is traceably linked to the appropriate raw data and all analysis and processing information. Once processed, the results are displayed in a user-defined graphical interface. In the plate view users can define two criteria to show the success, or otherwise, of the experiment. In Figure 3, the plate view shows information about the four concentrations at the end of the time course experiment. The left half displays information about the percentage of product with the correct 1:1 conjugation state (Green >50%, Yellow 10-50%, Orange 1-10% and Red 0%). The right-half shows the homogeneity of the sample, indicating which peaks in the sample are due to conjugated ralb (Green >90%, Orange 50-90% and Red 0-50%). As can be seen, all samples are still primarily conjugated ralb at the end of the time course, however for all samples less than 50% of the final product was a 1-to-1 ralb conjugate and both the 10:1 and 20:1 had no 1-to-1 conjugate remaining. Also produced is a user-defined results table in three formats: HTML, PDF and Excel. This table can be configured to include all critical results parameters, for example information regarding the conjugation state of the sample at each time point (i.e. average number of conjugations), the homogeneity of the sample (% intensity Tray view showing the conjugation results of F5M fraction of all conjugates) and the elapsed time of each sample. The results table for the time course experiment for ralb:f5m conjugation at a 1:1 molar ratio is shown in Figure 4. Each column can have a colour scheme set to determine whether desired user defined outcomes are met. In this example, for the experiment to be deemed successful the average number of conjugates must be between 0.5 and 1.5, the most abundant conjugate state must equal 1 and the % of the intensity due to conjugated ralb must be over 90%. For each sample analyzed an individual PDF document is produced, including sample and processing information, the TIC and deconvoluted spectra, and the deconvolution information (Figure 5). These PDFs can be collated into one document and attached to any related reports. The PDF documents are also of high value for manual quality control of the results, any unexpected results highlighted in the graphical interface can be quickly checked to decide whether the raw data needs to be further interrogated. Due to all processed results being produced in Excel format, further analysis is a straight-forward task. As an example, in Figure 6 we show the percentage of correct conjugation state (one ralb per conjugate) and the average number of conjugates per molecule of ralb, plotted against time after the conjugation reaction was started. The results indicate that at all concentration ratios, after an initial period of rapid conjugation, a relatively steady state is quickly reached. The greater the excess of conjugate the quicker the conjugation reaction proceeds, and the higher the number of conjugates that are seen on the molecule. Conclusion Biopharma Compass allows fully automated data acquisition, processing and analysis for mass spectrometric samples. Here we have shown that it can be used to monitor protein conjugation either for quality control purposes or, when running time course experiments, to determine optimum conjugation conditions. High throughput workflows can be easily run with minimal user intervention, allowing greater throughput and more accurate data analysis, while freeing staff for more productive and complex tasks. Figure 3: The tray view showing the conjugation results for the four concentrations of F5M:rAlb (from left, 1:1, 5:1, 10:1, 20:1) at the end of the time course experiment. A different color scheme has been used for each half of the wells in order to visually summarize the results of the experiment. The left half indicates the percentage of product with the correct 1:1 conjugation state (Green >50%, Yellow 10-50%, Orange 1-10% and Red 0%). The right half reports the homogeneity of the sample, indicating which peaks in the sample are due to conjugated ralb (Green >90%, Orange 50-90% and Red 0-50%).

5 Result summary table Sample Unmodified Protein Mr Max. No. of Conj. Mean No. of conjugates attached Most Abundant conjugation state Mr Base Peak [Da] Int. Frac. Base Peak [%] % Correct Conj. No. Purity (% Conjugates in TIC) Elapsed time(s) ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: ralb:f5m 1: Figure 4: Results summary table for the time course experiment for ralb:f5m conjugation at a 1:1 ratio. Three parameters were used to judge the success of the experiment, which are automatically shown in green if the criteria for success were fulfilled. In this example, the average number of conjugates must be between 0.5 and 1.5, the most abundant conjugate state must equal 1 and the purity of ralb and conjugates of ralb must be over 90%. Summary PDF report Figure 5: Automatically generated summary PDF report showing key information for how the data was acquired and processed. The total ion chromatogram is shown in addition to the deconvoluted spectrum and resulting mass peak list which contains the observed and identified conjugates.

6 Bruker Daltonics is continually improving its products and reserves the right to change specifications without notice. Bruker Daltonics , LCMS-87, Percentage of correct conjugation state 70 % Correct Conjugation :1 5:1 10:1 20:1 0 t/[s] 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 Average No. of Conjugants t/[s] 1:1 5:1 10:1 20:1 Figure 6: The percentage of correct conjugation state (one ralb per conjugate) and the average number of conjugates per molecule of ralb, plotted against time after the conjugation reaction was started. Data can be exported directly from BioPharma Compass to Excel, allowing easy interpretation of the experiment. References [1] Bruker Daltonics Application-Note ET-20 BioPharma Compass: A fully Automated Solution for Characterization and QC of Intact and Digested Proteins [2] Andersen, J. T. and Sandlie, I., The Versatile MHC Class I-related FcRn Protects IgG and Albumin from Degradation: Implications for Development of New Diagnostics and Therapeutics. Drug Metabolism and Pharmacokinetics, Vol. 24 (2009) No. 4, For research use only. Not for use in diagnostic procedures. Bruker Daltonik GmbH Bremen Germany Phone +49 (0) Fax +49 (0) sales@bdal.de Bruker Daltonics Inc. Billerica, MA USA Phone +1 (978) Fax +1 (978) ms-sales@bdal.com Fremont, CA USA Phone +1 (510) Fax +1 (510) cam-sales@bruker.com