Raman Spectroscopic Monitoring and Control of Aprotinin Supersaturation in Hanging-Drop Crystallization

Rosana E. Tamagawa, Everson A. Miranda, Kris A. Berglund

Research output: Contribution to journalArticle

  • 12 Citations

Abstract

Fiber optic Raman spectroscopy is used for in situ monitoring of supersaturation during the hanging-drop crystallization of aprotinin. Schwartz and Berglund (1999) previously demonstrated this technique for lysozyme crystallization and showed it combines two critical elements for protein crystallization studies: real-time monitoring/ control of supersaturation and small amounts of sample. Experiments were carried out using 10 μL of protein solution. A partial-least-squares (PLS) calibration based on Raman spectra of standard solutions allowed an accurate measurement of aprotinin in a range of 2-100 mg/mL with a standard error of 0.54 mg/mL determined by a leave-one-out cross validation. A 10x microscope attached to a Raman fiber optic probe allowed the monitoring of the hanging-drop liquid phase in a noninvasive and real-time mode. Aprotinin solubility determined by measuring the protein concentration of drop solution at equilibrium decreased with increase in NaCl concentration. By continuously collecting Raman spectra of the liquid phase in the drop, the protein concentration was monitored in real time during the whole process. Control of supersaturation by manipulating the evaporation rate of the drop solution allowed the optimization of the process, leading to an increase in the resulting crystal size.

LanguageEnglish (US)
Pages263-267
Number of pages5
JournalCrystal Growth and Design
Volume2
Issue number4
DOIs
StatePublished - Jul 2002

Profile

Aprotinin
Supersaturation
Crystallization
supersaturation
crystallization
proteins
Monitoring
Proteins
fiber optics
liquid phases
Fiber optics
Raman scattering
Raman spectra
evaporation rate
lysozyme
Liquids
Muramidase
solubility
Raman spectroscopy
microscopes

ASJC Scopus subject areas

  • Chemistry(all)
  • Materials Science(all)
  • Condensed Matter Physics

Cite this

Raman Spectroscopic Monitoring and Control of Aprotinin Supersaturation in Hanging-Drop Crystallization. / Tamagawa, Rosana E.; Miranda, Everson A.; Berglund, Kris A.

In: Crystal Growth and Design, Vol. 2, No. 4, 07.2002, p. 263-267.

Research output: Contribution to journalArticle

@article{93c0f5ec52c44acd849bb6bcf44312b7,
title = "Raman Spectroscopic Monitoring and Control of Aprotinin Supersaturation in Hanging-Drop Crystallization",
abstract = "Fiber optic Raman spectroscopy is used for in situ monitoring of supersaturation during the hanging-drop crystallization of aprotinin. Schwartz and Berglund (1999) previously demonstrated this technique for lysozyme crystallization and showed it combines two critical elements for protein crystallization studies: real-time monitoring/ control of supersaturation and small amounts of sample. Experiments were carried out using 10 μL of protein solution. A partial-least-squares (PLS) calibration based on Raman spectra of standard solutions allowed an accurate measurement of aprotinin in a range of 2-100 mg/mL with a standard error of 0.54 mg/mL determined by a leave-one-out cross validation. A 10x microscope attached to a Raman fiber optic probe allowed the monitoring of the hanging-drop liquid phase in a noninvasive and real-time mode. Aprotinin solubility determined by measuring the protein concentration of drop solution at equilibrium decreased with increase in NaCl concentration. By continuously collecting Raman spectra of the liquid phase in the drop, the protein concentration was monitored in real time during the whole process. Control of supersaturation by manipulating the evaporation rate of the drop solution allowed the optimization of the process, leading to an increase in the resulting crystal size.",
author = "Tamagawa, {Rosana E.} and Miranda, {Everson A.} and Berglund, {Kris A.}",
year = "2002",
month = "7",
doi = "10.1021/cg025524k",
language = "English (US)",
volume = "2",
pages = "263--267",
journal = "Crystal Growth and Design",
issn = "1528-7483",
publisher = "American Chemical Society",
number = "4",

}

TY - JOUR

T1 - Raman Spectroscopic Monitoring and Control of Aprotinin Supersaturation in Hanging-Drop Crystallization

AU - Tamagawa,Rosana E.

AU - Miranda,Everson A.

AU - Berglund,Kris A.

PY - 2002/7

Y1 - 2002/7

N2 - Fiber optic Raman spectroscopy is used for in situ monitoring of supersaturation during the hanging-drop crystallization of aprotinin. Schwartz and Berglund (1999) previously demonstrated this technique for lysozyme crystallization and showed it combines two critical elements for protein crystallization studies: real-time monitoring/ control of supersaturation and small amounts of sample. Experiments were carried out using 10 μL of protein solution. A partial-least-squares (PLS) calibration based on Raman spectra of standard solutions allowed an accurate measurement of aprotinin in a range of 2-100 mg/mL with a standard error of 0.54 mg/mL determined by a leave-one-out cross validation. A 10x microscope attached to a Raman fiber optic probe allowed the monitoring of the hanging-drop liquid phase in a noninvasive and real-time mode. Aprotinin solubility determined by measuring the protein concentration of drop solution at equilibrium decreased with increase in NaCl concentration. By continuously collecting Raman spectra of the liquid phase in the drop, the protein concentration was monitored in real time during the whole process. Control of supersaturation by manipulating the evaporation rate of the drop solution allowed the optimization of the process, leading to an increase in the resulting crystal size.

AB - Fiber optic Raman spectroscopy is used for in situ monitoring of supersaturation during the hanging-drop crystallization of aprotinin. Schwartz and Berglund (1999) previously demonstrated this technique for lysozyme crystallization and showed it combines two critical elements for protein crystallization studies: real-time monitoring/ control of supersaturation and small amounts of sample. Experiments were carried out using 10 μL of protein solution. A partial-least-squares (PLS) calibration based on Raman spectra of standard solutions allowed an accurate measurement of aprotinin in a range of 2-100 mg/mL with a standard error of 0.54 mg/mL determined by a leave-one-out cross validation. A 10x microscope attached to a Raman fiber optic probe allowed the monitoring of the hanging-drop liquid phase in a noninvasive and real-time mode. Aprotinin solubility determined by measuring the protein concentration of drop solution at equilibrium decreased with increase in NaCl concentration. By continuously collecting Raman spectra of the liquid phase in the drop, the protein concentration was monitored in real time during the whole process. Control of supersaturation by manipulating the evaporation rate of the drop solution allowed the optimization of the process, leading to an increase in the resulting crystal size.

UR - http://www.scopus.com/inward/record.url?scp=0346677856&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0346677856&partnerID=8YFLogxK

U2 - 10.1021/cg025524k

DO - 10.1021/cg025524k

M3 - Article

VL - 2

SP - 263

EP - 267

JO - Crystal Growth and Design

T2 - Crystal Growth and Design

JF - Crystal Growth and Design

SN - 1528-7483

IS - 4

ER -