ASTM E1470-92(2006)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E1470 − 92 (Reapproved2006)
Standard Test Method for
Characterization of Proteins by Electrophoretic Mobility
This standard is issued under the fixed designation E1470; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3. Significance and Use
1.1 This test method describes a procedure for determining 3.1 The prime purpose of this test method is to provide data
the electrophoretic mobility of proteins of molecular weight expressed as either electrophoretic mobility or zeta potential
greater than 10 000 Daltons. distribution of protein particles.
1.2 This test method uses automatic Electrophoretic Light 3.2 Both sellers and purchasers of protein particles will find
Scattering (ELS) principles to determine the electrophoretic this test method useful to determine either mobility or zeta
mobility. potentialdistributionsforproteinspecifications, manufacturing
2 control, and development and research.
1.3 The instrument simultaneously measures the Doppler
shifts of scattered light at four different angles to determine the
4. Apparatus
electrophoretic mobility distribution of protein particles. The
4.1 The apparatus for analysis consists essentially of a laser
mobility is expressed as µm-cm/V-s (micron-centimeter/volt-
light source, sample cell for introducing the sample, power
second).
supply source, four 256-channel spectrum analyzers,
1.4 This standard does not purport to address all of the
microprocessors, and computer assembly.
safety concerns, if any, associated with its use. It is the
4.2 Sample chamber assembly, holds approximately 1 mL
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica- of sample and is composed of three basic parts. The two side
pieces are made of solid silver and contain hemispherical
bility of regulatory limitations prior to use.
cavities. Between the two side pieces is a fused silica glass
2. Summary of Test Method
insert, running through it is a rectangular channel (3 mm wide
by 1 mm high). The channel connects the two cavities. Fluid
2.1 A carefully dispersed, dilute suspension of the protein
fills both cavities and the channel. Electrophoretic Light
particles is loaded into the sample cell and is positioned in the
Scattering measurements are made on particles in the channel.
path of collimated laser light. The laser light directed onto
4.2.1 30 mL Plastic Accuvetts,(disposable)forpreparingthe
particles moving at constant velocity under an applied electri-
sample.
cal field. The laser light is scattered from moving particles,
4.2.2 Membrane Filtering Device, 0.2 µm filters or finer.
producing a Doppler shift proportional to the particle’s veloc-
4.2.3 5 mL Sterile Plastic Syringe.
ity.
4.2.4 8 Gage Blunt Tipped Hypodermic Needle.
2.2 The instrument response is essentially to a sinusoidal
4.2.5 pH Meter.
“beat” signal produced at the detector by mixing the scattered
4.2.6 Standard Buffer Solution.
light and a reference (unscattered) beam. The frequency of the
“beat” signal is equal to the difference Doppler shift and
5. Reagents and Materials
therefore, to particle speed and direction.
5.1 Purity of Reagents—Reagent grade chemicals shall be
used in all tests. Unless otherwise indicated, it is intended that
1 all reagents shall conform to the specifications of the Commit-
This test method is under the jurisdiction of ASTM Committee E55 on
Manufacture of Pharmaceutical Products and is the direct responsibility of Subcom- tee on Analytical Reagents of the American Chemical Society
mittee E55.04 on General Biopharmaceutical Standards.
where specifications are available. Other grades may be used,
Current edition approved Nov. 1, 2006. Published December 2006. Originally
approved in 1992. Last previous edition approved in 1998 as E1470 – 92 (1998).
DOI: 10.1520/E1470-92R06.
2 3
The sole source of supply of the instrument (Coulter Delsa 440, trademark) Reagent Chemicals, American Chemical Society Specifications, American
known to the committee at this time is Coulter Corporation, 601 W. Coulter Way, Chemical Society, Washington, DC. For Suggestions on the testing of reagents not
Hialeah, FL 33010. If you are aware of alternative suppliers, please provide this listed by the American Chemical Society, see Annual Standards for Laboratory
information to ASTM International Headquarters. Your comments will receive Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
careful consideration at a meeting of the responsible technical committee, which and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
you may attend. MD.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1470 − 92 (2006)
provided it is first ascertained that the reagent is of sufficiently 7.1.1 Description of various systems.
high purity to permit its use without lessening the accuracy of 7.1.2 Description on limitations on electrophoretic mobility
the determination. range, particle size range, measured conductivity range, an
...