Zetasizer ZS Helix (DLS and Raman) for Bioph - Sysmex · Develop mechanistic insight into oligomerization and aggregation ... generate reports without making “expert level” ...

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

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 directlycomparable

“Expertise Optional” software analysis mode –generate reports without making “expert level”decisions

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

DLS Basics – Rayleigh Scattering

› Scattered light as a function of timetells us about average particle size Smaller particles – intensity correlated

over short time

Larger particles – intensity correlatedover longer time

› Particle Size Distribution

› Polydispersity Indicative of oligomers/aggregation

› Protein-protein interactions kD and B22

Change of signal with time correlates toparticles size distribution

Raman Basics – Inelastic Scattering

› Raman spectra give information aboutvibrations of molecular bonds

› Changes in the local environment ofthe protein backbone and amino acidresidues appear as changes to theRaman spectra a-helix and b-sheet content derived from

Amide I and III peaks – secondary structure

Hydrogen bonding extent, the localenvironment (hydrophilic or hydrophobic),dihedral angle, and other informationdescribing the local environment of tyrosineand tryptophan side chains appears inmultiple distinct peaks throughout thespectrum – tertiary structure

Disulfide bond conformations and quantityappear in a distinct region of the spectra –tertiary structure

From:https://depts.washington.edu/ntuf/facility/docs/NTUF-Raman-Tutorial.pdfDaniel Schwartz, Univ. of Washington, Dept. of Chem. Eng.

Raman Basics

› NOT Rayleigh scattering – but inelastic scatterEach band below corresponds to a specific protein backbone or side

chain bond, and its specific environment defined by its secondary andtertiary structure

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Raman Characterization

› Protein secondary and tertiary structurecharacterization 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

Fiber coupled Raman probe

Attenuator

Laser – 633 nm

Correlator

LensMovable mirror

Detector

Fiber coupler

Cuvette holder

RamanSpectrometer/Laser

– 785 nm

Schematic of ZS Helix Experiment

Raman and DLS datacollected in aninterleaved fashion

› Practicality of combining Liquid samples

Cuvette sampling

Fiber optic coupling

Limited moving parts

Modes of Operation

› Kinetics / Isothermal Incubation / Temperature Jump Determine kinetics of oligomerization and/or aggregation events

Investigate reversibility of size and structural changes duringthese processes

› Thermodynamics / Temperature Ramp Determination of thermodynamic properties

• Tonset, Tmelt

• Enthalphy/coopertivity of transitions

Compare relative stability of samples

Mechanistic understanding of oligomerization and aggregationevents

› Sample Series Evaluate impact of a perturbation across a range of samples

Perturbation other than temperature (pH, denaturant, etc.)

“Expertise Optional” Analysis Mode:Key Raman metrics pre-defined and automaticallycalculated

• Secondary structure – determined via multivariate model linking Raman and CD• Tertiary structure – moiety specific band analysis

“Expertise Optional” Analysis Mode:Automatically create parameter trend graphs

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

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 concentrationsamples – do these characterizations predict high concentrationbehavior???

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 proteininteractions that lead to aggregation (B22 + kD)

• Thermodynamics –Tonset, Tmelt, 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?

Raman/DLS Combined – Address Pain?

Provide mechanistic information• Aggregation• Unfolding/denaturation• Oligomerization

Improve product stability and formulation

Biopharmaceutical Application ExamplemAb and modified mAb (with dual variable domain)

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 andmmAb, 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 B22 and kD 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?

DLS/Raman Combined to Answer the Question:“What is happening at the low temp transition?”Compare to DSC collected on different instrument

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Temperature [C]

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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 stableTyrosine H-bonding environment mostperturbed 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

More Information on Kinetics of OligomerizationIsothermal incubation of mmAb at 46, 53, 60 °C

• 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 856.8 to 855.5 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 onetransition…

TryptophanTyrosineSize

ZS Helix Provides InsightsImprove product stability and formulation

› Story from DLS/Raman results:

› “… low temp transition is oligomerization event involvingtyrosine side chain – specifically showing up in the hydrogenbonding environment for this moiety.”

› “…high temp transition is a mass aggregation event.”

› “…oligomers formed at lot temp transition in mmAb arestable and irreversible.”

› Result: Tyrosine side chains can be manipulated to prevent lowtemp oligomerization and provide more stable mmAb

› What was missed previously using: SLS, DLS, DSC, DSF

› What is actually happening at that low temp transition?

Potential Application – Characterization ofNano-Milled API

› Use Ramanspectroscopy to IDand quantifypolymorph content

› DLS to determineparticle sizedistribution

From: Understanding Infrared and Raman Spectra of PharmaceuticalPolymorphs – Donahue et al, Am. Pharm. Rev., 4(2), 2011.