Optim 2

1. Optim 2 An Introduction

2. Avacta Analytical • Based in the UK with network of global distributers. • Leeds University in the North of England. • Exponential growth over the past 6 years through both natural growth and acquisition. • Currently around 80 full time employees.

3. The Challenge

4. Three points Specifically looking at protein stability • The need for speed • Low sample availability – especially in early development • The need for more information

5. Optim 2 Optim 2 High-throughput micro-volume characterisation of protein stability

6. Quick Overview • Combines fluorescence and light scattering into one instrument • Simultaneous investigation of conformational unfolding and aggregation propensity • Expressed as the thermal midpoint (Tm) and aggregation onset (Tagg) • All samples temperature controlled allowing for thermal ramping • Low sample volumes9µl • High throughput measurements 96 sample per day • Proprietary software with powerful data analysis

7. Application areas Preformulation Development Solubility, stress testing, clone (candidate) selection, early formulation development Formulation Development Stability, excipient studies. Further product characterisation. Process Development Explore reaction space and process variables to optimize yield and stability Anywhere you need to screen molecules or formulations for stability

8. Instrumental configuration

9. Illustrative pre-formulation screen Scenario – Pre-formulation study Objective – investigate physical stability of different formulations. One candidate under multiple conditions. Matrix of 3 buffers, 5 pH’s, 3 salt concentrations and 10 excipients measured all in triplicate 810 samples in total Illustrative case of Optim vs. other label free instruments

10. Illustrative pre-formulation screening Optim delivers in two working weeks results that would otherwise take 4 months to complete Notes: 1. 1mg of therapeutic antibody costs ~£1000 2. labour cost calculated at £20/hr 3. Sample requirement and instrument usage time data for the DSC and LS measurements were taken from manufacturer’s specifications where available 4. Malvern Zetasizer and Microcal DSC 5. Malvern Zetasizer APS and Microcal Capillary DSC 6. Additional cost reflects cost of Optim consumables 7. Total comes from 2030h for DSC & 1620h for DLS 8. Total comes from 390 for DSC & 1620 for DLS 9. Total comes from £16,200 DSC & £8,100 DLS

11. De-risk your development process A three month pre-formulation project (one month data collection a two months data analysis and interpretation). Here we compare the amount of samples you can screen using classical techniques, automated techniques and Optim

12. Using intrinsic protein fluorescence to measure protein conformational stability

13. Intrinsic fluorescence to measure protein conformation Tryptophan Tyrosine Phenylalanine UV light • Illuminate protein with UV light • Aromatic residues fluoresce – mainly tryptophan • Aromatic residues hydrophobic and buried away from water in folded protein

14. Intrinsic fluorescence to measure protein conformation • Intensity, peak wavelength and shape of spectrum depends on environment around fluorescent residues

15. Intrinsic fluorescence to measure protein conformation • When the protein unfolds partially or completely the environment around the fluorescing residues changes • The fluorescence spectrum changes in response

16. Change in florescence with temperature We can apply a temperature ramp and observe changes in intrinsic fluorescence Tm

17. Vary pH to observe effect on thermal stability pH 6.5 pH 4.5 pH 3.5 pH 2.5 Identify observed midpoint of thermal unfolding transition – Tm Higher Tm – higher conformational stability Vary solution parameters and observe effect on thermal stability

18. Static Light Scattering to measure protein aggregation propensity

19. Static Light Scattering (SLS) to monitor protein aggregation • Illuminate sample with laser - measure scattered intensity at 90° • For small solute particles scattered intensity proportional to mean solute mass x concentration • Therefore scattered intensity increases with aggregation

20. Static Light Scattering (SLS) to monitor protein aggregation Optim static light scattering highly sensitive: Dependence of scattering intensity on IgG monomer concentration 473 nm light scattering for higher dynamic range 266 nm light scattering for high sensitivity Wavelength dependence of scattering ~ 1/λ4

21. Optim static light scattering highly sensitive Dependence of scattering intensity on mean solute mass 0.1 mg/ml

22. Using static light scattering to give an indication of the aggregation propensity Apply temperature ramp and observe aggregation pH 4.5 pH 6.5 Tagg pH 3.5 pH 2.5

23. Key features summary Data rich High throughput Low sample Volume

24. Data rich Provision of multiple stability indicating measurement helps users predict stability profile of their molecules, de-risking your development programme Optim can perform simultaneous measurements Measuring intrinsic fluorescence shifts allowing the user to determine a thermal midpoint or Tm Light scattering enables the determination of the onset of protein aggregation or Tagg

25. UK Biopharmaceutical “The Optim 1000 is a data-rich method of analysis which uses very small amounts of material, for example, less than 320 mg for one entire study. This low sample requirement allowed us to rapidly screen a variety of different formulations, meaning that we could study even more than had previously been possible. Those formulations that were found to be unsuitable were discarded early in the development process, effectively de-risking the programme” Head of Preformulation, UK

26. Rapid and high throughput measurements Faster analysis allows you to increase the scope of your investigations Designed for speed 96 samples in one day - 48 samples in one run High performance imaging spectrograph is able to instantaneously acquire whole spectra measurements, quickly acquiring data

27. Low sample volume • Sample held in Micro Cuvette Array (MCA) • Specifically designed to give optimum florescence and light scattering signal from small sample volumes – 9µl <0.1 mg/ml to 150+ mg/ml (sample dependent) Low sample amounts enables more analytics to be completed earlier in the development process when sample availability is low. • Sealed for zero evaporation during heating • Spacing compatible with standard 384 well plate

28. University of Kansas Prof Russell Middaugh co-director, Center for Macromolecule and Vaccine Stabilization, University of Kansas. Published GEN, Sept 1st “Conventional analytical methods used for preformulation, stability, and formulation studies have previously relied on methods that extrapolate partial data on slow and labour-intensive instrumentation that is incompatible with high-throughput measurements and tight development timelines. The Optim 1000 microvolume protein analysis and characterization system offers rapid, multimodal analysis of ultra-low sample volumes at high throughputs.”

29. Summary • Optim helps reduce the risk of your drug development programme • Moreinformation, with less sample and in less time than conventional techniques