The Utility of H/DX-MS in Biopharmaceutical Comparability Studies

A presentation based on the research of Engen et al, which compares the utility of Hydrogen-Deuterium Exchange MS in Biopharmaceutics. HDX-MS is briefly introduced and some concepts are explained.

1. 1 The Utility of H/DX-MS in Biopharmaceutical Comparability Studies Individualized study unit Biopharmaceuticals: Protein Production and Analysis Abhijeet Lokras, Marija Fiodorovaite , Nikos Giannopoulos, Sven Hinca, Mereddy VJ Ragav D. Houde, S. A. Berkowitz, J. R. Engen

2. 2 Comparability studies Comparability Biopharmaceutical equivalence, absence of product changes 3D structure influences biological and physico-chemical properties Production relies on cells → less control than in synthesis PTMs in vivo and in vitro Comparability assessment Biochemical, biophysical, biological, clinical and nonclinical studies

3. 3 Comparability assessment: Challenge Variety of techniques commonly used: Circular dichroism (CD), Fluorescence, UV-spectroscopy Differential scanning calorimetry (DSC) Isothermal titration calorimetry (ITC) Analytical ultracentrifugation (AUC) Fourier transform infrared spectroscopy (FTIR) Dye binding Global readout → challenge in detecting small but significant changes Sophisticated methods (NMR, XRC) often can’t be used Method: too complex, time consuming Sample: too large or cannot be crystallized

4. 4 Comparability assessment: Answer H/DX-MS H/DX detected with NMR, FTIR Coupled with MS in 1991 Proteolytic digestion Peptide level (5-10 AA) Doesn’t determine protein structure, only conformational comparisons Low sample requirements Ability to measure protein regardless of size Native or formulated buffer form

5. Introduction to the H/DX technique The primary method for introducing deuterium into a protein sample is by dilution. Dilutions of 15-fold or greater will produce final deuterium concentrations of >95%. This serves to force the labelling reaction (k 2 ) in one direction [4] Labelling Method - Continuous Transition from a protiated species to one that is deuterated is unidirectional (under certain conditions) Figure 1: Process of H/D Exchange Figure 2: Global and Local difference Figure 3: Folding, Unfolding and deuteration

6. Back Exchange and Relative Deuterium Reuptake Back-exchange occurs because a) Proteolytic digestion b) Analysis of the deuterium levels done with protiated solvents Relative levels rather than absolute deuterium levels are used in this experiment. WHY? a) Does not require totally deuterated form of the molecule of interest. b) Sequence variation is no longer a factor (Same sequence is compared to itself) c) Variables such as slight changes in buffer pH, temperature, concentration are taken care of. The only thing – Experiments are to be performed at the same time, under identical conditions RFE = Dt/(Number of AA in peptide – 1 – Number of proline residues) Relative (Dt) = mavg,t – mavg,t0

7. Introduction to the Experiment GOALS: 1. Utility of H/DX-MS in biopharmaceutical industry 2. Higher order structures and structural dynamics of different preparations of Interferon β -1a (IFN) Total of 5 samples of IFN were used: a) Reference IFN (High Purity) b) IFN stored at -70 o C for over 8 years c) PEGylated IFN d) IFN made by different tissue culture medium and growth conditions e) Oxidized IFN (made from stored IFN) All IFN samples were at concentration 0.25 mg/mL in 100 mM PB with 200 mM NaCl pH=7.2

8. Scheme of the experimental process Figure 4: Scheme of the experimental process.

9. Results and discussion Four H/DX-MS comparisons: IFNref vs. IFNfrozen IFNref vs. PEG-IFN IFNref vs. IFN(dif. tissue and growth cond.) IFNref vs. IFNoxidized Methods: SEC, LC-MS, AUC, CD, disulfide mapping, fluorescence, UV. Results: all the samples were comparable , except IFNoxidized Figure 5. Biophysical characterization of native and oxidized IFN. A – SEC chromatogram, B – CD spectra, C – fluorescence spectra.

10. Preliminary assessment of comparability with H/DX-MS Figure 6. (a) Pepsin digestion coverage map, (b) LC TIC chromatogram (left) and MS spectra of peptide 1 (right) of H/DX-MS experiment. Figure 7. Selected deuterium incorporation graphs (right). Black – reference IFN, red – modified IFN. Left – the representation of the actual position of the selected peptides.

11. Alternative Modes to Analyze H/DX-MS Comparable Samples Comparability profile of two IFN samples based on different culture media and growth conditions: Each point is an average of four H/DX-MS comparison experiments Data acquired at 10s, 1, 10, 60 and 240 min of deuteration Critical analysis by measuring and plotting the corresponding differences D( Δ M i,t ) and D s ( i ) Limit lines showing 98% confidence that needs to be exceeded for two samples to be considered different Both samples are comparable Figure 8. Comparability profile of IFNref and IFNdif.cond .

12. Comparisons Based on one or two H/DX-MS Experiments Values for each of the four H/DX-MS experiments: D( Δ M i,t ) and D s ( i ) data that exceed the 98% confidence limits at low level and frequency Small DI(1) and DI(2) values When comparing reference IFN vs IFN stored at -70 °C for over 8 years: Values with more variability D s data exceed 98% confidence limits in a few areas DI values equal zero Similar results obtained when comparing reference IFN vs PEGylated IFN Figure 9. The average difference data.

13. Interday Comparisons Increased variability when the reference and experimental samples are analyzed on different days Noncomparable Samples Reference IFN vs oxidized IFN: Average of three H/DX-MS experiments Visual differences for peptides 6-7 and 30-35 Deuteration differences for peptides 29-38 and 48 Other smaller changes High DI(1) and DI(2) values Figure 10. Top – the effects of conducting the experiments on different days. Bottom graphs – comparability profile of IFNref . And IFNoxidized

14. Conclusions Challenges for protein comparability's in Bio-pharmaceutical drug development : H/DX-MS is not capable of analyzing conformational differences in coexisting low-level components of the population between two or more molecules. practical, high resolution, and routine analytical tools capable of providing the critical assessment of a protein’s spatial structure are significantly lacking. similar tools for assessing the temporal component of a protein’s three-dimensional structure are nearly nonexistent.

15. Future improvements for implementation of H/DX-MS into the biopharmaceutical industry eventual automation and seamless coupling of high-performance separation, robotic sampling, handling better MS methods and instrumental systems, and computer software aiming at H/DX-MS peptide identification, data analysis, and display. develop a complete commercial turn key H/DX-MS system. improve spatial resolution, and the use of gas-phase H/DX to obtain information on amino acid side chain behavior. Greater capability technology that can improve comparative, stability, formulation development and structure analysis of drug design.