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
So what was the motivation and rational behind the Optim 1000 development programme.
Through our knowledge of work as a CRO with the Biopharma market we know that decisions made early in development can have profound implications later on.

4. Three points Specifically looking at protein stability
The need for speed
Low sample availability – especially in early development
This limits the amount of information which can be collected about candidate molecules.
This can compromise the ability to make the best decisions early in 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 volumes 9µl
High throughput measurements 96 sample per day
Proprietary software with powerful data analysis
So I would like to start be running through a brief overview of the main points of what Optim is and what it can achieve.
Optim combines florescence and light scattering capabilities into one.
This allows the user to simultaneously investigate a proteins conformational unfolding and aggregation propensity.
We can express these two measurements as the thermal midpoint (or Tm) and aggregation onset temperature (or Tagg)
Optim has the ability to temperature ramp all the samples at the same time.
It is capable of using very low sample amounts.
Can take measurements very rapidly meaning it has high through put capabilities.
Finally all this is integrated with proprietary software and powerful data analysis tools.

7. Application areas
Discovery
Research
Lead Drug Identification
Lead Optimisation
Clinical Studies
GMP Manufacturing
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
So where can Optim be applied in the development process.
Well the answer is anywhere where you might want to screen molecules, formulations or different solvent conditions for stability.
To pick out a few examples:
Whilst in a preformulation screen, where you want to collect data on how your protein behaves in common solvent conditions.
In formulation where you are looking to identify the best formulation design space.
In process development whilst screening process variables to investigate manufacturability.

8. Instrumental configuration
So what is in the box that allows Optim to achieve all this.
We illuminate the sample with two laser – 266nm and 473nm.
The samples are presented in a micro cuvette - pictured in box, I will talk more about this later - which sits on a heating/cooling plate, which in turn is located on top of a precision XY positioning plate.
As the laser illuminates each sample in turn the imaging spectrograph simultaneously collects the scattered light data - at the two different wave lengths and the intrinsic florescence signal.
With this instrumental configuration we are able to quickly scan multiple samples whilst ramping up the temperature.
All this information is then collected, processed and presented in our data analysis software.

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
Protein Required (3)
Cost of Protein (1)
Instrument Time (3)
Total Man hours
Labour Cost (2)
Total Cost
Non-Automated - DSC/DLS 4
42 mg
$67,000
3650 h (7)
1215 h
$39,000 (9)
$106,000
Automated - HT-DSC/HT-DLS 5
12.2mg
$19,000
2010 h (8)
9.2 h.
$288
$19,288
Single instrument - Optim 2
0.73 mg
$1,170
74 h
8.4 h
$272
$3,369 (6)
Line one – Non Automated - Malvern Zetasizer Nano and Microcal DSC
Line two – Automated – Malvern Zetasizer APS and Microcal Capillary DSC
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
Examples
5.5, 6, 6.5, 7 4
Phosphate, Citrate, Acetate,Tris
NaCl at 0, 50, 150 mM 3
Tween 0, 0.01, 0.1 % v/v
Trehelose 0, 500 mM 2
Histidine 0, 50 mg/ml
Replicates
1728

12. Using intrinsic protein fluorescence to measure protein conformational stability
First I’d like to talk about the use of intrinsic protein fluorescence to monitor protein conformation.

13. Intrinsic fluorescence to measure protein conformation
Illuminate protein with UV light
Aromatic residues fluoresce – mainly tryptophan
Aromatic residues hydrophobic and buried away from water in folded protein
If we illuminate our protein with Ultraviolet light what we will find is that certain residues within the protein emit fluorescence.
Most of this florescence typically comes from the tryptophan residues.
The residues that fluoresce tend to be hydrophobic and thus when the protein is correctly folded they are buried away in its core, away from the water environment surrounding them in solution.
Graph – Here we have an example of a characteristic intrinsic spectrum with wave length along the x axis and intensity along the y axis.
You can see the characteristic peak of fluorescence intensity at 330 nm which is typical for a correctly folded protein.
Tryptophan
Tyrosine
Phenylalanine
UV light

14. Intrinsic fluorescence to measure protein conformation
Intensity, peak wavelength and shape of spectrum depends on environment around fluorescent residues
What we find is that if we change the environment surrounding the residues we can observe various changes in the intrinsic fluorescence spectra.
Graph - We can see changes in the intensity of the spectrum, so the height of the peak - Changes in the position of the peak, so the centre wave length here - Changes in the shape of the peak, so the width of the peak

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
We can see that if protein unfolds the residues, which were previously buried in the heart of the protein, become exposed to the solvent environment.
This change in environment results in a change of the fluorescent properties of the protein.
Graph – This is illustrated here with the spectra on the right. The blue spectrum is from a native protein and the red from a denatured protein.
In the native protein (blue) has a lower florescence intensity and the peak is around 330nm.
In the denatured protein (red) we can see an increased intensity and shift to a longer wave length.
We have further illustrated this by normalising the spectra, as you can see in the top right hand corner, the red spectra clearly shows a shift in wave length.

16. Change in florescence with temperature
We can apply a temperature ramp and observe changes in intrinsic fluorescence Tm
So we have demonstrated that we can use florescence to look at changes in conformation and in particular to monitor the unfolding of the proteins.
This enables us to monitor unfolding as we apply a temperature ramp.
Graph - Here we have an illustration of this, the x axis is the increase in temperature and the y is effectively the change in peak position.
We can clearly see a transition event here at 50 C, this can be termed as a thermal midpoint or Tm.

17. Vary pH to observe effect on thermal stability
Vary solution parameters and observe effect on thermal stability
pH 6.5
pH 4.5
We have demonstrated how we use Florescence to determine the unfolding temperature and we would expect that the higher the Tm the more conformationally stable the protein is.
What we can now do is change the solvent environment around the protein and see what effect that has on its conformational stability.
We illustrate this here, what we have done is take our protein, an IgG in this case and place it in a range of buffers at different pH values.
Graph - Here we can see that at a low pH of 2.5 – (dark blue line) – the protein has a much lower Tm – around the point. At the higher pH of 6.5 – (light blue line) – has a much higher Tm – around the high 60s – to 70.
Meaning that this protein has a better conformational stability around the higher pH.
pH 3.5
pH 2.5
Identify observed midpoint of thermal unfolding transition – Tm
Higher Tm – higher conformational 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
In the Optim system we illuminate the sample in a micro capillary with two lasers and collect the scattered light at an angle of 90 degrees.
What we find is that with the small solute particles – so very small aggregates – the intensity of the scattered light is proportional to the mean solute mass times by the concentration of the protein.
There for if we have a sample of a certain protein concentration the scattered intensity will increase with the average size of the aggregates in the sample.

20. Static Light Scattering (SLS) to monitor protein aggregation
Optim static light scattering highly sensitive: Dependence of scattering intensity on IgG monomer concentration
266 nm light scattering
for high sensitivity
473 nm light scattering
for higher dynamic range
Here we show that the static light scattering in Optim is very sensitive, in the left plot what we have is an increase in the concentration of an IgG from 0mg/ml up to 1mg/ml.
We can see a linear relationship, this shows that the light scattering is sensitive even to the monomer protein.
We have two wave lengths of light scattering that we operate with, 266nm and 473nm.
The 266 is very sensitive, and is able to detect very small aggregates - but can saturate if you get to much aggregation.
The 473 light scattering is slightly less sensitive but gives you a higher dynamic range so you can measure up to higher amount of aggregation before the signal saturates.
Wavelength dependence of scattering ~ 1/λ4

21. Optim static light scattering highly sensitive
Dependence of scattering intensity on mean solute mass
Again another illustration of how the static light scattering works, here what we have done is us some dextran size standards, and we have recorded the 266 scattering intensity from each of these.
You can see for a 1mg/ml solution there is a linear relationship between dextran size and scattering intensity.
In the smaller graph on the right we have shown that the same relationship exitists even at 0.1mg/ml
0.1 mg/ml

22. Using static light scattering to give an indication of the aggregation propensity
Apply temperature ramp and observe aggregation
So how do we use that light scattering in practise?
Previously shown how we can monitor fluorescence, well we can simultaneously monitor the light scattering as well.
In a typical experiment we can apply a temperature ramp to the sample.
In this plot we can see an example of what information you can gain from this. The first thing we can see is that this protein sample, which has been put in a range of solutions of varying pH, starts aggregating at different temperatures depending on the pH.
We define this onset of aggregation as Tagg or the onset of aggregation temperature.
The higher the aggregation onset temperature the more stable the protein is seen to be.
This combined with the simultaneous unfolding data can give you a strong insight into the stability of your protein whilst in varying solvent conditions.
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 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
Provision of multiple stability indicating measurement helps users predict stability profile of their molecules, de-risking your development programme
Optim is a data rich technique.
Not only are you able to get information on the thermal unfolding and aggregation onset of your protein.
You are also able to investigate unfolding rate, by holding temperature at a constant.
And you are able to look at protein-protein interaction.
The use of these multiple stability indicating measurements can feed into your decision process and enable you to make informed decisions on how you want to continue the development your therapeutic.
Helping to de-risk your development.

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
Here is another customers view.
Syntaxin used Optim for their preformulation screen.
It enabled them decrease there total sample amount requirements whilst at the same time investigating more formulations.
The end result being they de-risked their development process.

26. Rapid and high throughput measurements
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
Faster analysis allows you to increase the scope of your investigations
From the very outset Optim was designed for speed.
It is able to measure 48 samples in one run and 96 samples in one day.
The high performance spectrograph is able to instantly acquire the whole spectrum. This enables Optim to quickly collect the relevant sample information and move on to the next sample in less than a second.
The ability to do this kind of analysis in a high through put manner allows you the user to increase the scope of their investigation. All of a sudden you can increase your preformulation screen from looking at 10 different conditions to looking at 100.

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 mg/ml (sample dependent)
Low sample amounts enables more analytics to be completed earlier in the development process when sample availability is low.
What gives the Optim the ability to use such low volumes?
This can be partially dueto the two different techniques being employed by Optim (Florescence and light scattering).
But our sample presentation method also has a large part to play in the impressively low sample usage.
The micro Cuvette array (or MCA) has a row 16 small capillary microcuvettes embedded within.
It is specifically designed to give optimum florescence and light scattering signal from small sample volumes and uses high grade crystal quartz.
This enables Optim to use as little as 9uL per sample.
The sample are also sealed to prevent evaporation.
And the spacing is compatible with a 384 well plate.
This low sample usage means Optim gives its user the ability to perform stability screen 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.”
This is an extract from an article published in GEN by Professor Russ Middaugh from Kansas university.
He clearly identifies the advantages to him saying Optim “offers rapid, multimodal analysis of ultra-low sample volumes at high throughputs”

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