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CHIRASCAN SERIES APPLICATION NOTE

INTRODUCTION

Circular Dichroism (CD) is a key analytical technique used to provide essential information on Higher Order Structure (HOS) comparability of protein drugs for regulatory submissions. This can be in support of new innovative drug submission, as a process change validation, or as an important part of the characterisation of a potential biosimilar against an original innovator drug.

In this study a biosimilar manufacturer wished to verify that the HOS and thermal stability of their drug were similar to an already marketed innovator drug. To do this the Chirascan™-plus ACD spectrometer was used to make multiple replicate measurements of the innovator and biosimilar drug of far ultraviolet (FUV), near ultraviolet (NUV) and thermal denaturation experiments. The experiments confirmed similarity of the biosimilar to the innovator.

Author:BERNARD COSTELLO, PhD Senior Applications ScientistApplied Photophysics Ltd. KEYWORDS

Automated Circular Dichroism Pharmaceutical

Biosimilar Higher Order Structure

Thermal stability Statistical analysis Confidence interval g-factor

Abstract: A pharmaceutical manufacturer wished to verify that the higher order structure and thermal stability of their drug were similar to those of an already marketed innovator drug. Statistical analysis of measurements made on the Chirascan-plus automated circular dichroism (ACD) spectrometer combined with statistical analysis was used to establish that there was no excursion beyond 95% confidence intervals between the two drugs in either respect.

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Comparison of Innovator and Biosimilar Biotherapeutic Drugs using Automated

Circular Dichroism Spectroscopy

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EXPERIMENTAL

The innovator and biosimilar drug were exchanged into a 50mM pH 2 phosphate buffer at a nominal concentration of 0.8 mg/ml. The experimental conditions are shown in Table 1. In all cases, CD and absorbance spectra were measured simultaneously.

FUV spectroscopy NUV spectroscopy Thermal meltsWavelength

range

260 to 180 nm 330 to 250 nm 260 to 180 nm

Wavelength

interval

1 nm 0.5 nm 1 nm

Bandwidth 1 nm 1 nm 1 nmTime per point 1 second 1 second 0.8 secondsScan time

(approx.)

90 seconds 3 minutes 90 seconds

Pathlength 0.2 mm 10 mm 0.2 mmTemperature 20°C 20°C 20°C to ~82°CTemperature

ramp rate

- - 1°C per minute

For the isothermal procedures, 3 replicates were used with 5 repeats. For the thermal melt procedures 3 replicates were used with a single repeat. These conditions are summarised in Table 2.

Procedure Replicates Repeats Total spectra

Sample FUV 3 5 15

Buffer FUV 6 5 30

Sample NUV 3 5 15

Buffer NUV 6 5 30

Sample Tm 3 1 3

Buffer Tm - - -

For all three procedures, the loading scheme in the 96-well microplate was:

Buffer / Biosimilar Sample / Buffer / Innovator Sample / Buffer/ Biosimilar Sample / Buffer / Innovator Sample / Buffer / Biosimilar Sample / Buffer / Innovator Sample.

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Table 1. Summary of experimental

Table 2. Summary of experimental

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RESULTS

ISOTHERMAL MEASUREMENTS

The spectra are shown here after averaging and correction by subtraction of the buffer contribution. To remove the effect of concentration difference between the samples, the spectra are also shown normalised by absorbance; the Innovator spectra were multiplied by a normalisation coefficient calculated as the ratio of the area under the Biosimilar absorbance spectrum to the area under the Innovator absorbance spectrum, where the areas were calculated using the midpoint rule.

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Figure 1. FUV absorbance spectra, the Biosimilar (red) and Innovator (cyan); before (left) and after normalisation by area. The normalisation coefficient was 1.0808

Figure 2. CD spectra, the Biosimilar (red) and Innovator (cyan) after normalisation by absorbance;

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The average and standard deviation, σ of the replicates and repeats were taken, with the confidence interval calculated as 2 σ, equivalent to about 95% probability, where σ is given by:

where n is the number of measurements. The uncertainty propagated when one spectrum is subtracted from another was calculated as:

Propagated uncertainty = [(Uncertainty 1)2 + (Uncertainty 2)2 ]0.5

For this analysis to be meaningful, it is important that the data are unfiltered, i.e. that no real-time or post-experimental smoothing has been applied.

Data are shown below plotted as the difference spectra, i.e. the Biosimilar spectrum – normalised Innovator spectrum, with the propagated uncertainty intervals plotted as centred on zero. This way of presenting the data enables a rapid assessment of the similarity of the spectra to be made, since an excursion of the difference spectrum from the uncertainty interval bars implies a 95% probability of the spectra being dissimilar.

The data are also shown plotted as g-factor against wavelength for both samples. The g-factor is defined as ∆A/A where ∆A is the circular dichroism in absorbance units, and A is the absorbance. This is a useful alternative way of viewing the data, since it removes the effects of sample concentration and pathlength, but it is not meaningful where the CD and absorbance are very low, since the ratio becomes dominated by the measurement uncertainties.

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Figure 4. Difference CD spectrum with 2 σ confidence interval bars, FUV (left) and NUV

Figure 5. g-factor for the Biosimilar (red) and Innovator (cyan), FUV (left) and NUV (right);

2

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APL APPLICATION NOTE Comparison of Innovator and Biosimilar Biotherapeutic Drugs using Automated Circular Dichroism

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THERMAL MELT MEASUREMENT

Figure 5 shows the thermal melt data for the Biosimilar and the Innovator. Only replicate 1 is shown; for both proteins replicates 2 and 3 were similar to replicate 1.

Results were analysed using Applied Photophysics’ Global 3 analysis software. A single transition model was used, with single slope baseline correction. A summary of the results is shown in Table 3; the ± values are calculated as 2 σ

Replicate Biosimilar Innovator1 58.1 58.22 58.3 57.73 57.7 57.6

Average 58.0 ± 0.5 57.8 ± 0.5

DISCUSSION AND CONCLUSIONS

There is no excursion of either the different FUV or NUV CD spectrum beyond the 95% confidence interval bars, suggesting that to within this degree of confidence there are no differences between the FUV and NUV spectra of the Biosimilar and Innovator under the conditions used. The g-factor spectra support this observation. The averages of the three replicates of the thermal melts were also within 95% confidence limits.

The same methodology can be applied to other types of HOS characterisation for areas such as validation of process changes or for initial regulatory submission of innovator drugs. This can be combined with the power of the ACD automation to scale this type of study to much larger numbers of samples, taking into account multiple different batches or processes.

Figure 6. Replicate 1 thermal melts for the Biosimilar (left) and Innovator (right); arrows indicate direction of increasing temperature.

Table 3: results of Global 3 analysis of the thermal melt data

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