Zetasizer ZS Helix (DLS/Raman) For Characterization of Pharmaceuticals and Biopharmaceuticals

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1 Zetasizer ZS Helix (DLS/Raman) For Characterization of Pharmaceuticals and Biopharmaceuticals

2 Benefits of Zetasizer ZS Helix What it provides Develop mechanistic insight into oligomerization and aggregation Use that insight to improve biopharmacuetical product How it works Characterize particle size distribution And simultaneously Characterize protein secondary and tertiary structure Additional Benefits Same sample, same time/save sample, save time Same heating rate - DLS and Raman data directly comparable Expertise Optional software analysis mode generate reports without making expert level decisions

3 Overview of Presentation Dynamic light scattering (DLS) basics Raman basics Combination of DLS and Raman - benefits Experimental description Expertise Optional data analysis Biopharmaceutical application example Mechanistic insight into oligomerization and aggregation Small molecule pharmaceutical potential application Characterization of nano-milling

4 DLS Basics Rayleigh Scattering Scattered light as a function of time tells us about average particle size Smaller particles intensity correlated over short time Larger particles intensity correlated over longer time Particle Size Distribution Polydispersity Indicative of oligomers/aggregation Protein-protein interactions k D and B 22 Change of signal with time correlates to particles size distribution

5 Raman Basics Inelastic Scattering Raman spectra give information about vibrations of molecular bonds Changes in the local environment of the protein backbone and amino acid residues appear as changes to the Raman spectra a-helix and b-sheet content derived from Amide I and III peaks secondary structure Hydrogen bonding extent, the local environment (hydrophilic or hydrophobic), dihedral angle, and other information describing the local environment of tyrosine and tryptophan side chains appears in multiple distinct peaks throughout the spectrum tertiary structure Disulfide bond conformations and quantity appear in a distinct region of the spectra tertiary structure From: Raman-Tutorial.pdf Daniel Schwartz, Univ. of Washington, Dept. of Chem. Eng.

6 Raman Basics NOT Rayleigh scattering but inelastic scatter Each band below corresponds to a specific protein backbone or side chain bond, and its specific environment defined by its secondary and tertiary structure Cystine Phenyalanine Tyrosine Tryptophan Tyrosine Tryptophan Backbone Phenyalanine Tryptophan Tryptophan Tyrosine Normalized Raman Intensity , Amid e III Tryptophan Glutamate Backbone Tryptophan Side chains Amide I o Wavenumber (cm -1 )

7 Raman Characterization Protein secondary and tertiary structure characterization In-depth analysis of 2e and 3e structure Impact of this on stability of the molecule Chemical identification small molecule API Raman fingerprint can be compared to library spectra for chemical ID Raman spectra vary with polymorphic form

8 Schematic of ZS Helix Experiment Raman and DLS data collected in an interleaved fashion Attenuator Laser 633 nm Detector Fiber coupler Practicality of combining Liquid samples Correlator Cuvette sampling Fiber optic coupling Limited moving parts Movable mirror Fiber coupled Raman probe Lens Cuvette holder Raman Spectrometer/Laser 785 nm

9 Modes of Operation Kinetics / Isothermal Incubation / Temperature Jump Determine kinetics of oligomerization and/or aggregation events Investigate reversibility of size and structural changes during these processes Thermodynamics / Temperature Ramp Determination of thermodynamic properties T onset, T melt Enthalphy/coopertivity of transitions Compare relative stability of samples Mechanistic understanding of oligomerization and aggregation events Sample Series Evaluate impact of a perturbation across a range of samples Perturbation other than temperature (ph, denaturant, etc.)

10 Expertise Optional Analysis Mode: Key Raman metrics pre-defined and automatically calculated Secondary structure determined via multivariate model linking Raman and CD Tertiary structure moiety specific band analysis

11 Expertise Optional Analysis Mode: Automatically create parameter trend graphs

12 Expertise Optional Analysis Mode: Automatic fit of trends provide quantitative results

13 Biopharmaceutical Industry What they want from their drug product formulation Stable formulations Easy to administer customer delivered, not IV at hospital Efficacious Low volume High concentration Pain points resulting from what you want (above) Aggregation issues Highly viscous Solubility issues Current analytical techniques require low concentration samples do these characterizations predict high concentration behavior???

14 DLS/Raman for Biopharma Raman spectroscopy and DLS combined for the analysis of protein therapeutics (to start ) Provide insight into the protein structural changes that drive unfolding/denaturation/aggregation Protein size distribution, polydispersity, measure of protein interactions that lead to aggregation (B 22 + k D ) Thermodynamics T onset, T melt, and DH Kinetics rate constants of unfolding/structural changes For QbD/formulation development Compare relative formulation stability but beyond screening Is a transition caused by aggregation, oligomerization, or unfolding? Which side chains and what environmental conditions are changing?

15 Raman/DLS Combined Address Pain? Provide mechanistic information Aggregation Unfolding/denaturation Oligomerization Improve product stability and formulation

16 Biopharmaceutical Application Example mab and modified mab (with dual variable domain)

17 mmab Narrative prior to ZSHelix evaluation The mmab candidate is less stable than the mab high temp transition at ~70C is the same for both mab and mmab, but the aggregation pathway appears to be different But - mmab exhibits a low temp transition at ~53 C as well What is going on at the low temp transition? Analytical techniques used prior to Helix evaluation: SLS, DLS, DSC, DSF, susceptibility to denature at air-liquid interface, SDS-PAGE, accelerated and real-time stability Conclusions drawn prior to Helix evaluation: Low concentration B 22 and k D values NOT predictive of high concentration behavior Different aggregation pathways exist for mab and mmab Unanswered questions remaining, prior to Helix evaluation: What is happening at low temp transition? What are the different aggregation pathways?

18 DLS/Raman Combined to Answer the Question: What is happening at the low temp transition? Compare to DSC collected on different instrument tyrosine region Red DSC Red - DSC Green DLS Blue - Raman DLS Rh Temperature [C] Lewis, Patent Pending

19 Method Used to Answer Question: Compare thermal ramp behavior: mab vs mmab Raman results mmab double transition: ~54& 63 C mab single transition: ~ 69 C mab and mmab respond differently to the thermal stress, mmab less stable Tyrosine H-bonding environment most perturbed during low temperature transition Sizing results mmab double transition: ~53 & 65 C mab single transition: ~ 69 C PDI trend for mmab oligomerization followed by aggregation

20 More Information on Kinetics of Oligomerization Isothermal incubation of mmab at 46, 53, 60 C Size Tyrosine Tryptophan 46 C - pre-transition size slowly increases Raman maker bands show no change 53 C in-transition size quickly increases from 10 to ~35 nm and then stabilizes Tyr peak position drops, while the Trp peak position shifts slightly Tyr peak position shifts from to cm -1, the value seen at the first transition in the T ramps 60 C post-transition size quickly increases to over 40 nm within 3 hours Trp and Tyr peak positions quickly shift to lower frequencies Tyr peak position shifts to ~855.5 cm -1. Not shown here heat mmab over low T transition temperature, cool back down. Then heat up to 80 only one transition

21 ZS Helix Provides Insights Improve product stability and formulation Story from DLS/Raman results: low temp transition is oligomerization event involving tyrosine side chain specifically showing up in the hydrogen bonding environment for this moiety. high temp transition is a mass aggregation event. oligomers formed at lot temp transition in mmab are stable and irreversible. Result: Tyrosine side chains can be manipulated to prevent low temp oligomerization and provide more stable mmab What was missed previously using: SLS, DLS, DSC, DSF What is actually happening at that low temp transition?

22 Potential Application Characterization of Nano-Milled API Use Raman spectroscopy to ID and quantify polymorph content DLS to determine particle size distribution From: Understanding Infrared and Raman Spectra of Pharmaceutical Polymorphs Donahue et al, Am. Pharm. Rev., 4(2), 2011.