Breckenridge Protein Stability Conference - 2013

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Introduction Materials and Methods Results Spectroscopic Characterization of Protein Structure Dynamics in Aqueous States Yemin Xu 1 , Chris Carpenter 2 , Korben Knudson 3 , Rina Dukor 4 , John Carpenter 5 , Theodore Randolph 3 1 Biochemistry Department, University of Colorado Boulder 2 Dept. of Physics, University of Colorado Boulder 3 Dept. of Chemical and Biological Engineering, University of Colorado Boulder 4 BioTools, Inc 5 Dept. of Pharmaceutical Sciences, University of Colorado, Denver Maintaining protein high order structure integrity is critical for developing protein pharmaceuticals. Mis-folded or unfolded protein pharmaceuticals will compromise their efficacy, change the pharmacokinetics profile and/or cause severe immune-responses. Thus, it is important to develop fast and accurate methods to routinely monitor the proteins pharmaceutical’s structures. In this study, lysozyme was used as model protein, formulated at 50mg/ml and incubated at elevated temperatures (40 and 65 °C) for one week. Raman spectroscopy and Circular Dichroism (CD) were employed to measure the lysozyme structure in aqueous solution. Particle formation was monitored by Flowcam, and soluble aggregates were examined and quantified by size exclusion chromatography (SEC). The extent of structural perturbation observed during incubation exhibited good correlation with the rate of aggregation and sub- visible particle formation under accelerated conditions. The results demonstrate that Raman spectroscopy could be a quick and reliable way to predict protein stability and high order structure integrity during incubation. 50mg/mL Lysozyme (H2O) pH 4.0 Samples divided into two groups (40 o Cand 65 o C) 40 o C 65 o C Samples were placed in the incubators for measuring at various time points (0-Point, Day 1, Day 4 , and Day 7, respectively). At each time point, triplicate samples were removed from the incubator and measured by different methods. Lysozyme from chicken egg white (Sigma-L6876): 50mg/ml (H 2 O, pH 4.0) Incubation temperature: 40±2 and 65±2 o C Incubation time point: 0, 1, 4 and 7 days Protein particle measurement: Flowcam (0.2mL sample size) Soluble protein aggregation: SEC (mobile phase: 0.1 M Phosphate buffer 0.1 M Na 2 SO 4, pH 6.7 ) Non-Reducing SDS Gel Proteins structure measurement: CD (Far-UV 0.3mg/ml, Near-UV 50mg/ml) Raman (50mg/ml, laser power 100mW at 780nm ) 2013 Colorado Protein Stability Conference Discussion Fig 4: Protein secondary and tertiary structure measured by circular dichroism (CD) A) Far-UV: measuring secondary structure; B) near-UV: measuring tertiary structure. Each color curve is the average of triplicate result. Different colors show different samples incubated at different temperatures. CD data show similar secondary structures for all samples, however, tertiary structures measured by near-UV indicate protein tertiary strcutures of samples incubated at 65 o C differ from others after 4 days. Each spectra represents average of triplicate measurements. A B A Disulfide bond S-S C Amide III B Tyrosine D Amide I Fig 1: Protein structure measured by Raman Center panel is the entire spectra of 50mg/ml lysozyme in aqueous solution (control) and 80 o C for 20 hours. Close look (A, B, C, D)of each representative region of disulfide bond, tyrosine, amide III and amide I indicates that protein structures were significantly perturbed after storing at high temperature (80 o C). A Disulfide bond S-S C Amide III B Tyrosine D Amide I Fig 2: Raman spectra of proteins incubated for various times at 40 o C Center panel is the entire spectra of 50mg/ml lysozyme at 40 o C for different time points Close look (A, B, C, D)of each representative region of disulfide bond, tyrosine, amide III and amide I indicates that protein structures were similar after storing at 40 o C for 1 week. Each spectra represents average of triplicate measurements. A Disulfide bond S-S C Amide III B Tyrosine D Amide I Fig 3: Raman spectra of proteins incubated for various times at 60 o C Center panel is the entire spectra of 50mg/ml lysozyme at 60 o C for different time points Close look (A, B, C, D)of each representative region of disulfide bond, tyrosine, amide III and amide I indicates that protein structures were disrupted after 4 days storing at 60 o C. Each spectra represents average of triplicate measurements. Fig 5: percentage of remaining monomer measured by SEC Results of triplicate measurements show that no noticeable protein loss was seen for samples incubated at 40 o C for one week, in contrast, there was around 10% monomer lysozyme lost after one week at 65 o C. Fig 6: non-reducing SDS gel (coomassie blue stain) Non-reducing SDS gel shows that there are noticeable aggregates from samples incubated at 65 o C after 4 days. MW kD 50 37 25 20 15 10 Fig 7: Sub-visible particle concentration measured by flowcam Within the flowcam measurable size range, protein particle size and concentration increase for both 40 o C and 60 o C samples. Representative images from each size range are shown Conclusions References Structure information from Raman spectroscopy and CD measurement show that there are structure perturbations in samples incubated at 65 o C after 4 days. Particle concentration decreased after 4 days, it may due to the size of protein aggregates is beyond the capable range of flowcam Far-UV measurement did not pick up noticeable differences between samples, but near-UV did, which indicates that under experiment condition, lysozyme likely undergoes tertiary structural perturbation. Raman spectroscopy is a useful tool to examine the protein high order structure, and it is capable to identify the structural differences occurring at various position (eg. Disulfide bond, tyrosine, amide III and amide I region).) It is critical and helpful to use orthogonal methodologies to evaluate protein stability. 1. R. S. Porubcan, K. L. Watters and J. T. McFarland, Arch. Biochem. Biophys., 1978, 186, 255-264. 2.J. L. Lippert, D. Tyminski and P. J. Desmeules, Journal of the American Chemical Society, 1976, 98, 7075-7080. 3.A. T. Tu, Raman spectroscopy in biology : principles and applications, Wiley, New York, 1982. 4.Z.-Q. Wen, J. Pharm. Sci., 2007, 96, 2861-2878.

Transcript of Breckenridge Protein Stability Conference - 2013

Page 1: Breckenridge Protein Stability Conference - 2013

Introduction

Materials and Methods

Results

Spectroscopic Characterization of Protein Structure Dynamics in Aqueous States

Yemin Xu1, Chris Carpenter2, Korben Knudson3, Rina Dukor4, John Carpenter5, Theodore Randolph3

1Biochemistry Department, University of Colorado Boulder 2Dept. of Physics, University of Colorado Boulder

3Dept. of Chemical and Biological Engineering, University of Colorado Boulder4BioTools, Inc

5Dept. of Pharmaceutical Sciences, University of Colorado, Denver

Maintaining protein high order structure integrity is critical fordeveloping protein pharmaceuticals. Mis-folded or unfolded proteinpharmaceuticals will compromise their efficacy, change thepharmacokinetics profile and/or cause severe immune-responses.Thus, it is important to develop fast and accurate methods toroutinely monitor the proteins pharmaceutical’s structures.

In this study, lysozyme was used as model protein, formulated at50mg/ml and incubated at elevated temperatures (40 and 65 °C) forone week. Raman spectroscopy and Circular Dichroism (CD) wereemployed to measure the lysozyme structure in aqueous solution.Particle formation was monitored by Flowcam, and solubleaggregates were examined and quantified by size exclusionchromatography (SEC).

The extent of structural perturbation observed during incubationexhibited good correlation with the rate of aggregation and sub-visible particle formation under accelerated conditions. The resultsdemonstrate that Raman spectroscopy could be a quick and reliableway to predict protein stability and high order structure integrityduring incubation.

50mg/mL Lysozyme (H2O)

pH 4.0

Samples divided into two groups (40oCand 65oC)

40oC 65oC

Samples were placed in the incubators for measuring at various time points (0-Point, Day 1, Day 4 , and Day 7, respectively).

At each time point, triplicate samples were removed from the incubator and measured by different methods.

Lysozyme from chicken egg white (Sigma-L6876):50mg/ml (H2O, pH 4.0)

Incubation temperature:40±2 and 65±2oC

Incubation time point: 0, 1, 4 and 7 days

Protein particle measurement: Flowcam (0.2mL sample size)

Soluble protein aggregation: SEC (mobile phase: 0.1 M Phosphate buffer 0.1 M Na2SO4, pH 6.7 ) Non-Reducing SDS Gel

Proteins structure measurement: CD (Far-UV 0.3mg/ml, Near-UV 50mg/ml) Raman (50mg/ml, laser power 100mW at 780nm)

2013 Colorado Protein Stability Conference

Discussion

Fig 4: Protein secondary and tertiary structure measured by circular dichroism (CD)A) Far-UV: measuring secondary structure; B) near-UV: measuring tertiary structure.Each color curve is the average of triplicate result. Different colors show different samples incubated at different temperatures.CD data show similar secondary structures for all samples, however, tertiary structures measured by near-UV indicate proteintertiary strcutures of samples incubated at 65oC differ from others after 4 days. Each spectra represents average of triplicatemeasurements.

A B

A

Disulfide bond S-S

C

Amide III

B

Tyrosine

D

Amide I

Fig 1: Protein structure measured by RamanCenter panel is the entire spectra of 50mg/ml lysozyme in aqueous solution (control) and 80oC for 20 hours.Close look (A, B, C, D)of each representative region of disulfide bond, tyrosine, amide III and amide I indicates that proteinstructures were significantly perturbed after storing at high temperature (80oC).

A

Disulfide bond S-S

C

Amide III

B

Tyrosine

D

Amide I

Fig 2: Raman spectra of proteins incubated for various times at 40oCCenter panel is the entire spectra of 50mg/ml lysozyme at 40oC for different time pointsClose look (A, B, C, D)of each representative region of disulfide bond, tyrosine, amide III and amide I indicates that proteinstructures were similar after storing at 40oC for 1 week. Each spectra represents average of triplicate measurements.

A

Disulfide bond S-S

C

Amide III

B

Tyrosine

D

Amide I

Fig 3: Raman spectra of proteins incubated for various times at 60oCCenter panel is the entire spectra of 50mg/ml lysozyme at 60oC for different time pointsClose look (A, B, C, D)of each representative region of disulfide bond, tyrosine, amide III and amide I indicates that proteinstructures were disrupted after 4 days storing at 60oC. Each spectra represents average of triplicate measurements.

Fig 5: percentage of remaining monomer measured by SECResults of triplicate measurements show that nonoticeable protein loss was seen for samples incubated at40oC for one week, in contrast, there was around 10%monomer lysozyme lost after one week at 65oC.

Fig 6: non-reducing SDS gel (coomassie blue stain)Non-reducing SDS gel shows that there are noticeableaggregates from samples incubated at 65oC after 4 days.

MW kD

50

37

25201510

Fig 7: Sub-visible particle concentration measured by flowcamWithin the flowcam measurable size range, protein particle size andconcentration increase for both 40oC and 60oC samples. Representativeimages from each size range are shown

Conclusions References

Structure information from Raman spectroscopy and CD measurement show that there are structure perturbations in samples incubated at 65oC after 4 days.

Particle concentration decreased after 4 days, it may due to the size of protein aggregates is beyond the capable range of flowcam

Far-UV measurement did not pick up noticeable differences between samples, but near-UV did, which indicates that under experiment condition, lysozyme likely undergoes tertiary structural perturbation.

Raman spectroscopy is a useful tool to examine the protein high order structure, and it is capable to identify the structural differences occurring at various position (eg. Disulfide bond, tyrosine, amide III and amide I region).)

It is critical and helpful to use orthogonal methodologies to evaluate protein stability.

1. R. S. Porubcan, K. L. Watters and J. T. McFarland, Arch. Biochem. Biophys., 1978, 186, 255-264.

2.J. L. Lippert, D. Tyminski and P. J. Desmeules, Journal of the American Chemical Society, 1976, 98, 7075-7080.

3.A. T. Tu, Raman spectroscopy in biology : principles and applications, Wiley, New York, 1982.

4.Z.-Q. Wen, J. Pharm. Sci., 2007, 96, 2861-2878.