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Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography
Published on: September 29, 2011
Protein chromatography on hydroxyapatite columns.
Larry J Cummings1, Mark A Snyder, Kimberly Brisack
1Bio-Rad Laboratories, Inc., Hercules, California, USA.
Methods in Enzymology
|November 7, 2009
Summary
Spherical hydroxyapatite chromatography enables efficient protein purification, reducing impurities like host cell proteins and DNA. This method enhances protein load capacity and column longevity for biopharmaceutical applications.
Area of Science:
- Biochemistry
- Chemical Engineering
- Chromatography
Background:
- Spherical hydroxyapatite offers advantages for protein scientists in purification.
- Biopharmaceutical companies utilize single and multiple column applications for complex samples.
Purpose of the Study:
- To review the use of spherical hydroxyapatite in protein purification.
- To discuss adsorption/desorption mechanisms and column lifetime factors.
- To cover column packing methods and diverse applications.
Main Methods:
- Review of adsorption and desorption mechanisms of hydroxyapatite.
- Analysis of chemical interactions affecting column lifetime.
- Evaluation of column packing methods for spherical and microcrystalline hydroxyapatite.
Main Results:
- Multiple column purification allows higher protein loads and better impurity reduction.
- Hydroxyapatite interactions with ions, metals, and phosphates impact column longevity.
- Low ionic strength buffers can cause significant pH shifts due to hydroxonium ion dynamics.
Conclusions:
- Spherical hydroxyapatite is a versatile tool for protein purification, particularly for recombinant proteins and monoclonal antibodies.
- Understanding hydroxyapatite's multimodal properties is key to optimizing purification strategies.
- Robust packing methods and buffer selection are crucial for maximizing column performance and lifespan.
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