SECOND DERIVATIVE UV FOR RAPID CONFORMATIONAL ASSESSMENT OF PROTEINS

NOVEMBER 2016 KEVIN MCCOWEN SUPERVISOR ANALYTICAL DEVELOPMENT

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OVERVIEW

• Brief overview of UV absorbance

• Development of statistical analysis to perform

comparative studies

• Instrumentation overview

• Introduction to Althea

• Applications of the method in the Althea Analytical Development lab

UV Absorbance, Very Brief Theory

Phenylalanine Tyrosine Tryptophan

• UV absorbance is due to the absorbance of photons by the pi electrons of aromatic rings

• Each of the amino acids with a ring structure absorb in the near UV area of the light spectrum at slightly different wavelengths

• These absorbance minima and maxima are sensitive to the microenvironment in the protein making this technique sensitive to conformational shifts in a protein

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ADVANTAGES OF CONVERSION OF UV ABSORBANCE SPECTRA TO 2ND DERIVATIVE

Increased Resolution

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-0.8

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250 260 270 280 290 300

Baseline Correction

The zero order absorbance profile of all proteins looks very similar-converting to second derivative resolves subtle variations

Second derivative calculation is independent of signal magnitude, conversion results in baseline correction and normalization

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METHODS OF CONVERSION TO 2ND DERIVATIVE • Can be obtained by optical and electronic techniques but in general these methods

have been replaced by mathematical modeling

• For this study we used an adaptive degree polynomial filter* which gives greater freedom in the polynomial order used to convert the data to adjust to changing noise around the different peaks

*Barak, Anal. Chem. 1995, 67, 2758-2762

Savitzky Golay Calculated ADPF Calculated

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LIMITATIONS OF VISUAL ASSESSMENT OF 2ND DERIVATIVE UV

• Visual comparison can be misleading, two spectra of the same protein can appear different while spectra of proteins with slight modifications to conformation can appear similar by the same visual criteria

• A method needed to be implemented to remove the subjectivity and to automate the method

Overlay of two injections of the same protein

Overlay of a native and denatured protein

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INSTRUMENTATION OVERVIEW

UHPLC:Thermo Scientific™ Vanquish™ Horizon UHPLC System Detectors: DAD and FLD

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RETENTION TIME PRECISION

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SIGNAL REPRODUCIBILITY OF THE VANQUISH DAD

n=100 Max 8.76 Mean 2.85

100 injections X 600 data points with the average RSD less than 3%, including the noise

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USING THERMO SCIENTIFIC™ CHROMELEON™ CDS TO COMPARE SPECTRA

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SPECTRAL CORRELATION OF BSA IN PBS

λ=250-290 N=50

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SPECTRAL CORRELATION OF DIFFERENT SERUM ALBUMINS COMPARED TO BSA

SSA=sheep serum albumin HS-A= human serum albumin RSA=rabbit serum albumin

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BSA DENATURED IN ORGANIC SOLVENTS

• Slight changes in conformation, as were expected with 5% and 10% acetonitrile, didn’t show a marked lack of correlation in these plots

• This led us to explore the use of a sliding correlation coefficient scheme to look at correlations in narrow regions of the spectra

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SLIDING CORRELATION COEFFICIENTS

0.016752 0.016353 0.999476 0.016418 0.01603 0.016418 0.01603 0.999199 0.016063 0.015696 0.016063 0.015696 0.016063 0.015696 0.998961 0.015697 0.015355 0.015697 0.015355 0.015697 0.015355 0.015697 0.015355 0.998816

0.01533 0.015012 0.01533 0.015012 0.01533 0.015012 0.01533 0.015012 0.01533 0.015012 0.998795 0.014963 0.01467 0.014963 0.01467 0.014963 0.01467 0.014963 0.01467 0.014963 0.01467 0.014595 0.014328 0.014595 0.014328 0.014595 0.014328 0.014595 0.014328 0.014595 0.014328 0.014228 0.013986 0.014228 0.013986 0.014228 0.013986 0.014228 0.013986 0.014228 0.013986 0.013862 0.013645 0.013862 0.013645 0.013862 0.013645 0.013862 0.013645 0.013862 0.013645 0.013507 0.013308 0.013507 0.013308 0.013507 0.013308 0.013507 0.013308 0.013507 0.013308 0.013171 0.012979 0.013171 0.012979 0.013171 0.012979 0.013171 0.012979 0.013171 0.012979 0.012858 0.012662 0.012858 0.012662 0.012858 0.012662 0.012858 0.012662 0.012858 0.012662 0.012565 0.012352 0.012565 0.012352 0.012565 0.012352 0.012565 0.012352 0.012565 0.012352 0.012283 0.012048 0.012283 0.012048 0.012283 0.012048 0.012283 0.012048 0.012283 0.012048 0.012002 0.011744 0.012002 0.011744 0.012002 0.011744 0.012002 0.011744 0.012002 0.011744 0.011721 0.01144 0.011721 0.01144 0.011721 0.01144 0.011721 0.01144 0.011721 0.01144

0.01144 0.011136 0.01144 0.011136 0.01144 0.011136 0.01144 0.011136 0.011159 0.010832 0.011159 0.010832 0.011159 0.010832

0.01088 0.010531 0.01088 0.010531 0.010608 0.010241

0.01035 0.009975 0.010109 0.009737 0.009882 0.009522 0.009663 0.00932 0.009445 0.00912 0.009227 0.008919 0.009009 0.008719 0.008791 0.008519 0.008575 0.00832 0.008371 0.008135 0.008188 0.007973

Protein1 Protein2

Second Derivative

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USING A SLIDING CORRELATION COEFFICIENT TO DETECT CONFORMATIONAL DIFFERENCES IN DENATURING SOLUTIONS OF BSA

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250 260 270 280 290

Rsq of BSA in PBS vs. Acetonitrile

vs. 5% ACN

vs. 10% ACN

vs. 15% ACN

vs. 20% ACN

vs. 25% ACN

vs. 30% ACN

Avg. Rsq Vs. 5% ACN 0.9957 Vs. 10% ACN 0.9935 Vs. 15% ACN 0.9920 Vs. 20% ACN 0.9917 Vs. 25% ACN 0.9743 Vs. 30% ACN 0.8635

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CORRELATION COEFFICIENT OF 4 DIFFERENT SERUM ALBUMINS

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250 255 260 265 270 275 280 285 290

BSA vs. SSA

SSA vs. HSA

BSA vs. HS-A

SSA vs. RSA

BSA vs. RSA

-0.01

-0.005

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250 260 270 280 290

BSA

SSA

HSA

RSA

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275 280 285 290

BSA VS. SSA

• Plotting the correlation coefficients shows obvious lack of correlation even between BSA and SSA which showed higher correlation values using a full scan correlation coefficient

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ALTHEA OVERVIEW

• Full service contract manufacturing organization (CMO) • Analytical development (early phase-commercial manufacturing support) • Formulations (proprietary Crystalomics technology) • State of the art Antibody Drug Conjugate facility (ADC)

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ALTHEA ANALYTICAL DEVELOPMENT AND FORMULATIONS/CRYSTALOMICS

• Anaytical Development • Supports external and internal clients (manufacturing, process

development, Formulations/Crystalomics) • Works with clients in early phase development through commercial

manufacturing

• Formulations/Crystalomics • Crystalomics is a proprietary technology that enables packaging of

high concentration proteins in a low viscosity formulation (infusions become injectables)

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HOW IS SECOND DERIVATIVE UV ANALYSIS BEING IMPLEMENTED AT ALTHEA

• Early phase analytics • Characterization of reference standard • Can be used for rapid in-process analysis to verify consistent protein

conformation • Can be used to support method development (protein peak ID, peak

purity) • Used in formulations to determine consistent conformation and rapidly

assess different formulation/crystalization conditions

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IgG IN PBS VS. pH2.5 AND 6M GUANIDINE

-0.015

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260 270 280 290

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260 270 280 290

IgG in 1X PBS

IgG at pH 2.5

Overlay of IgG in 1X PBS

Overlay of IgG in 1X PBS vs. pH 2.5

Curves appear very similar visually, but the correlation coefficient, as well as other comparitive methods, failed to show the similarity with high confidence

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EVALUATION USING A SLIDING SCALE CORRELATION COEFFICIENT

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250 260 270 280 290 300

IgG in 1X PBS

IgG in 6M Guanidine

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IgG in 1X PBS

IgG in pH 2.5

Blue trace represents the Rsq of duplicate injections of IgG in 1X PBS

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ASSESSING CRYSTALIZATION CONDITIONS

Chromeleon Spectral Library Match

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ASSESSING CRYSTALIZATION CONDITIONS

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CONCLUSIONS • 2nd derivative UV analysis can provide a sensitive method for detection of

conformational changes in proteins

• This method is proving useful for rapid analysis of intermediates, comparative studies of proteins, and rapidly assessing formulation conditions

• Utilizing the method requires some knowledge of reproducibility of the comparator protein

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ACKNOWLEDGEMENTS Ajinomoto Althea • Austin Jackson

• Maria Green

• Lena Ceballos

Thermo Scientific • Jeanine Pippitt