ASTM E1759-95(2003)
(Test Method)Standard Test Method for Isoaspartic Acid in Proteins: Method for the Determination of Asparagine Deamidation Products (Withdrawn 2003)
Standard Test Method for Isoaspartic Acid in Proteins: Method for the Determination of Asparagine Deamidation Products (Withdrawn 2003)
SIGNIFICANCE AND USE
Isoaspartic acid residues are generated during incubation of proteins under a wide variety of conditions in aqueous solution. Such residues are generated most commonly through the deamidation of aspargine residues although some reports of isoaspartic acid formation through the rearrangement of aspartic acid residues have been published.
The presence of such residues can indicate that the protein containing such residues has suffered damage that may affect the biological activity of the protein. The precise correlation between the level of isoaspartic acid content and the biological activity of the protein needs to be determined on a case by case basis.
The test measures the level of isoaspartic acid content in a protein sample. This level will often be correlated with the degree to which the protein has suffered deamidation at asparagine residues. In addition, isoaspartic acid residues can arise on occasion through the rearrangement of aspartic acid residues. For these reasons, the level of isoaspartic acid residues in proteins can be used as a general indication that the protein sample has suffered some level of damage and should not be interpreted to indicate the precise level of damage to any one region within a protein without further testing.
SCOPE
1.1 This test method covers the determination of isoaspartic acid residues in a protein or peptide sample. This test method is applicable for the determination of isoaspartic acid residues in a sample in the range of 2.5-50 μmol/L. Higher concentrations can be determined following dilution. The reported lower range is based on single-operator precision.
1.2 The values stated in SI units are to be regarded as the standard.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
WITHDRAWN RATIONALE
This test method covers the determination of isoaspartic acid residues in a protein or peptide sample.
Formerly under the jurisdiction of Committee E55 on Manufacture of Pharmaceutical Products, this test method was withdrawn in July 2012 in accordance with section 10.5.3.1 of the Regulations Governing ASTM Technical Committees, which requires that standards shall be updated by the end of the eighth year since the last approval date.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:E1759–95 (Reapproved 2003)
Standard Test Method for
Isoaspartic Acid in Proteins: Method for the Determination
of Asparagine Deamidation Products
This standard is issued under the fixed designation E1759; 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.
INTRODUCTION
The storage of proteins in aqueous solutions often results in the formation of isoaspartic acid
linkages within the polypeptide chain as a result of the deamidation of aspargine residues and the
rearrangement of aspartic acid linkages. This test measures the amount of isoaspartic acid residues in
a protein or peptide solution by the use of the enzyme protein isoaspartyl methyl transferase and
radioactive S-adenosyl-L-methionine.
1. Scope and radiolabelled S-adenosyl-L-methionine, a radiolabelled
form of a co-factor that is consumed in the enzymatic reaction
1.1 This test method covers the determination of isoaspartic
of the enzyme. During the test a radiolabelled intermediate is
acid residues in a protein or peptide sample. This test method
formed through the transfer of the labeled methyl group from
is applicable for the determination of isoaspartic acid residues
S-adenosyl-L-methionine to the alpha carboxy group of isoas-
in a sample in the range of 2.5–50 µmol/L. Higher concentra-
partic acid. This methylated intermediate is then degraded to
tions can be determined following dilution. The reported lower
liberate the methyl group as methanol. The methanol is then
range is based on single-operator precision.
captured in a methanol diffusion procedure and counted.
1.2 The values stated in SI units are to be regarded as the
3.2 A sample of protein is incubated with the enzyme
standard.
protein isoaspartyl methyl transferase and radiolabelled
1.3 This standard does not purport to address all of the
S-Adenosyl Methionine in a buffer that results in the accumu-
safety concerns, if any, associated with its use. It is the
lation of the methyl esters of isoaspartic acid residues through
responsibility of the user of this standard to establish appro-
the enzymatic transfer of the methyl group from S-adenosyl-
priate safety and health practices and determine the applica-
L-methionine to isoaspartic acid sites in the protein. The
bility of regulatory limitations prior to use.
protein solution is then treated with a basic solution containing
2. Terminology sodium dodecyl sulfate in order to inactivate the enzyme and
convert the methylated isoaspartic acid residues to a succin-
2.1 Definitions of Terms Specific to This Standard:
imide and free methanol. The methanol is then separated from
2.1.1 isoaspartic acid residue—indicates an aspartic acid
the protein solution through the diffusion of the methanol to a
residue in which linkage of the polypeptide chain takes place
scintillation fluid solution. The methanol transferred to the
through the gamma carboxyl group of the aspartic acid versus
scintillation fluid is then determined by counting of the
the alpha carboxyl group that is used in the normal peptide
radioactivity in the scintillation fluid.
linkage.
4. Significance and Use
3. Summary of Test Method
4.1 Isoaspartic acid residues are generated during incuba-
3.1 The basis of the procedure given in this test method is
tion of proteins under a wide variety of conditions in aqueous
the production of radioactive methanol equal to the amount of
solution. Such residues are generated most commonly through
isoaspartic acid residues present in a protein sample through
the deamidation of aspargine residues although some reports of
the action of the enzyme protein isoaspartyl methyl transferase
isoaspartic acid formation through the rearrangement of aspar-
tic acid residues have been published.
This test method is under the jurisdiction of ASTM Committee E55 on
4.2 The presence of such residues can indicate that the
Manufacture of Pharmaceutical Products and is the direct responsibility of Subcom-
protein containing such residues has suffered damage that may
mittee E55.04 on General Biopharmaceutical Standards.
affect the biological activity of the protein. The precise
Current edition approved April 10, 2003. Published April 2003. Originally
approved in 1995. Last previous edition approved in 1995 as E1759 – 95. DOI:
10.1520/E1759-95R03.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1759–95 (2003)
correlationbetweenthelevelofisoasparticacidcontentandthe 8. Calibration
biological activity of the protein needs to be determined on a
8.1 Prepare 50 pmol/5 mLreference standard solution and a
case by case basis.
reactionblanksolution(0pmol/5mL).DilutetheIsoAsp-DSIP
4.3 The test measures the level of isoaspartic acid content in
reagentprovidedwiththeISOQUANT kitwithwatertocreate
a protein sample. This level will often be correlated with the
the reference standard solution and use the water for the
degree to which the protein has suffered deamidation at
reaction blank solution.
asparagine residues. In addition, isoaspartic acid residues can
9. Procedure
arise on occasion through the rearrangement of aspartic acid
residues. For these reasons, the level of isoaspartic acid
9.1 Determinethenumberofreactionsthatwillberuninthe
residues in proteins can be used as a general indication that the
test. Each test should contain a 0 and 50 pmol IsoAsp-DSIP
protein sample has suffered some level of damage and should
standard along with any unknowns. All samples and standards
notbeinterpretedtoindicatethepreciselevelofdamagetoany
are to be used in duplicate specimens.
one region within a protein without further testing.
9.2 Prepare a IsoAsp-DSIP reference standard by diluting
the IsoAsp-DSIP standard to 10 µmol in a 1.5 mL microcen-
5. Interfering Substances
trifuge tube with water and mixing by vortex for 15 s. Prepare
5.1 Sodium dodecyl sulfate and guanidine hydrochloride
at least 20 µL of diluted reference standard. Refer to the
will interfere with this test by inactivating the enzyme. certificate of analysis provided with the IsoAsp-DSIP material
5.2 Highly acidic, basic or buffered solutions that alter the
for the exact concentration of the standard with the kit to be
pH of the reaction mixture from pH 6.2 can interfere with the used.
assay by altering the kinetics of the enzymatic reaction used in
9.3 Calculate the amount of H-SAM stock solution needed
the test in either a positive or negative way. in the assay. For each reaction to be run, add 1.1 µL of
S-adenosyl-L-methionine and 1.1 µCi of H-SAM to a 1.5 mL
6. Apparatus
microcentrifugetubeandaddwatertoafinalvolumeof11mL.
9.4 Preparereactionmastermix.Foreachreactiontoberun,
6.1 Scintillation Counter.
add 11 mL of water; 11 mL of 5X reaction buffer; 11 mL of
6.2 Scintillation Vials—Scintillation vials capable of hold-
protein isoaspartyl methyltransferase; and 11 mL of H-SAM
ing at least 4.5 mL of scintillation fluid and capable of being
stock solution in a 1.5 mL microcentrifuge tube. Add the
heated to 40°C for an extended period of time without damage
materials in the order given and mix by vortex 15 s.
in the presence of scintillation fluid are used.
9.5 Place two labeled 1.5 mL microcentrifuge tubes on ice
6.3 Microcentrifuge.
for the reaction blank, the 50 pmol IsoAsp-DSIP calibration
6.4 Positive Displacement Pipettes.
standard and for each sample to be run.
7. Reagents and Materials 9.6 Insert one sponge insert into a scintillation vial cap for
2 every reaction that will be performed.Attach the sponge insert
7.1 Protein Isoaspartyl Methyltransferase.
2 to the inside of the vial cap by removing the backing on the
7.2 IsoAsp-DSIP (Delta Sleep Inducing Peptide).
2 sponge and attaching the adhesive site of the sponge to the
7.3 5X Reaction Buffer.
2 inside of the cap.
7.4 S-Adenosyl-L-Methionine.
2 9.7 Fill a scintillation vial to half its capacity with scintil-
7.5 Stop Solution.
2 lation fluid for each assay to be performed.
7.6 Sponge Inserts.
3 9.8 Add 10.0 mL of each unknown and reaction blank
7.7 Tritiated S-Adenosyl-L-Methionine, [ H-SAM].
sample to the appropriate labeled sample tube and place the
7.8 Scintillation Cocktail—A standard scintillation fluid
tubeonice.Add5.0mLoftheIsoAsp-DSIPreferencestandard
with a flash point greater than or equal to 150°C and capable of
and 5.0 mL of water to each reference standard sample tube
use for the counting of
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