SECOND DERIVATIVE UV FOR RAPID CONFORMATIONAL ASSESSMENT OF PROTEINS

Transcription

1 SECOND DERIVATIVE UV FOR RAPID CONFORMATIONAL ASSESSMENT OF PROTEINS NOVEMBER 2016 KEVIN MCCOWEN SUPERVISOR ANALYTICAL DEVELOPMENT

2 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 1

3 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

4 ADVANTAGES OF CONVERSION OF UV ABSORBANCE SPECTRA TO 2 ND DERIVATIVE Increased Resolution 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 3

5 METHODS OF CONVERSION TO 2 ND DERIVATIVE Can be obtained by optical and electronic techniques but in general these methods have been replaced by mathematical modeling For this study we used an adaptive degree polynomial filter* which gives greater freedom in the polynomial order used to convert the data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, Savitzky Golay Calculated ADPF Calculated 4

6 LIMITATIONS OF VISUAL ASSESSMENT OF 2 ND DERIVATIVE UV Overlay of two injections of the same protein Overlay of a native and denatured protein 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 5

7 INSTRUMENTATION OVERVIEW UHPLC:Thermo Scientific Vanquish Horizon UHPLC System Detectors: DAD and FLD 6

8 RETENTION TIME PRECISION 7

9 SIGNAL REPRODUCIBILITY OF THE VANQUISH DAD 100 injections X 600 data points with the average RSD less than 3%, including the noise n=100 Max 8.76 Mean

10 USING THERMO SCIENTIFIC CHROMELEON CDS TO COMPARE SPECTRA 9

11 SPECTRAL CORRELATION OF BSA IN PBS λ= N=50 10

12 SPECTRAL CORRELATION OF DIFFERENT SERUM ALBUMINS COMPARED TO BSA SSA=sheep serum albumin HS-A= human serum albumin RSA=rabbit serum albumin 11

13 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 12

14 SLIDING CORRELATION COEFFICIENTS Second Derivative Protein1 Protein

15 USING A SLIDING CORRELATION COEFFICIENT TO DETECT CONFORMATIONAL DIFFERENCES IN DENATURING SOLUTIONS OF BSA 1.2 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 Vs. 10% ACN Vs. 15% ACN Vs. 20% ACN Vs. 25% ACN Vs. 30% ACN

16 CORRELATION COEFFICIENT OF 4 DIFFERENT SERUM ALBUMINS BSA vs. SSA SSA vs. HSA BSA vs. HS-A SSA vs. RSA BSA vs. RSA BSA SSA HSA RSA 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

17 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) 16

18 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) 17

19 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 18

20 IgG IN PBS VS. ph2.5 AND 6M GUANIDINE Overlay of IgG in 1X PBS Curves appear very similar visually, but the correlation coefficient, as well as other comparitive methods, failed to show the similarity with high confidence Overlay of IgG in 1X PBS vs. ph IgG in 1X PBS IgG at ph 2.5

21 EVALUATION USING A SLIDING SCALE CORRELATION COEFFICIENT Blue trace represents the Rsq of duplicate injections of IgG in 1X PBS IgG in 1X PBS IgG in ph IgG in 1X PBS IgG in 6M Guanidine

22 ASSESSING CRYSTALIZATION CONDITIONS Chromeleon Spectral Library Match 21

23 ASSESSING CRYSTALIZATION CONDITIONS 22

24 CONCLUSIONS 2 nd 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 23

25 ACKNOWLEDGEMENTS Ajinomoto Althea Austin Jackson Maria Green Lena Ceballos Thermo Scientific Jeanine Pippitt 24