EP0423312A1 - Detection of endotoxin in pharmaceutical preparations by mass spectrometry - Google Patents
Detection of endotoxin in pharmaceutical preparations by mass spectrometryInfo
- Publication number
- EP0423312A1 EP0423312A1 EP90907628A EP90907628A EP0423312A1 EP 0423312 A1 EP0423312 A1 EP 0423312A1 EP 90907628 A EP90907628 A EP 90907628A EP 90907628 A EP90907628 A EP 90907628A EP 0423312 A1 EP0423312 A1 EP 0423312A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- hydrolysate
- endotoxin
- beta
- alcohol
- mass spectrometry
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Definitions
- the present invention relates to a method of detection of endotoxin in polypeptide compositions. More specifically, this invention relates to the measurement of endotoxin obtained by ⁇ uantitating the amount of a volatile derivatized component (beta-hydroxymyristic acid) of endotoxin present in a sample of any polypeptide. compositions.
- the present invention can be used for evaluating endotoxin content of products which are intended for human and veterinary use as parenteral therapeutic agents in clinical medicine, including products produced by recombinant DNA technology.
- Polypeptides produced by recombinant DNA technology have only recently become available' for use as therapeutic agents in clinical medicine.
- Polypeptides such as human insulin, human growth hormone, alpha-interferon and tissue plasminogen activator have all been approved by the Food and Drug Administration (FDA) for the treatment of human diseases.
- FDA Food and Drug Administration
- Several other interferons such as alphainterferon, beta-interferon, and subsets of gammainterferon
- interleukin-2 and tumor necrosis factor are now in early clinical stages of trial at centers throughout the world as treatments for cancer.
- Many polypeptide factors are expected to be manufactured and tested clinically over the next several years.
- the present invention establishes a more efficient method of measuring endotoxin, by production and detection of a volatile derivatized component unique to endotoxin.
- the present invention relates to A Method for detection of endotoxin in a polypeptide composition
- a Method for detection of endotoxin in a polypeptide composition comprising the steps of: hydrolysis of the composition containing said endotoxin, to form beta-hydroxymyristic acid; esterifying the resulting hydrolysate with an alkanol; reducing the carbalkoxy groups present in the hydrolysate to hydroxymethyl groups; separating the volatile
- the invention further relates to a method for detection of endotoxin in a polypeptide composition comprising the steps:
- hydrolysis of the polypeptide composition esterification of the resulting hydrolysate with an alkanol; acylation of free amino groups present in the hydrolysate; reduction of the carbalkoxy groups in the hydrolysate to hydroxymethyl groups; extraction of beta-hydroxymyristyl alcohol from the reduced hydrolysate; derivation of the extracted alcohol; separating the volatile derivatized product from the hydrolysate; and detecting and quantitating the volatile derivatized alcohol of beta-hydroxymyristic acid by mass spectrometry.
- the present invention contemplates a method of detection of the presence of endotoxin in a polypeptide composition.
- endotoxin as used in the specification and claims also includes gram-negative bacterial pyrogen and lipopolysaccharide (LPS).
- LPS lipopolysaccharide
- the method of the present invention involves converting endotoxin by hydrolysis into beta-hydroxymyristic acid, which is subsequently converted into beta- hydroxymyristyl alcohol, which can be detected by mass spectrometry after separation from ether hydrolysis products.
- the method of producing the volatile derivative of beta-hydroxymyristic acid can be accomplished by the steps of hydrolyzing the biological system containing endotoxin; esterification of the resulting hydrolysate; conversion of carbalkoxy groups present in the hydrolysate by reduction to hydroxymethyl (-CH 2 OH) groups and separation of the volatile alcohols from the hydrolysate.
- a preferred method of producing the volatile derivative of beta-hydroxymyristic acid can be accomplished by the following steps:
- step (e) derivatizing the extracted alcohol of step (e), preferably by trimethylsilylation of the free alcohol groups.
- the silylated product can then be separated using known means, preferably by gas chromatcgraphy, although column chromatcgraphy may also be used.
- the volatile derivatized component can then be identified and quantified by mass spectrometry techniques.
- Reduction of carbalkoxy groups to hydroxymethyl groups is preferably carried out by using a borohydride reducing reagent.
- amino groups of the hydrolysate if present, will complex with the boron of the reducing reagent.
- this step is selectively accomplished by the choice of suitable solvents such as haiogenated hydrocarbons, e.g., methylene chloride, ethylene chloride, dichloroethane and the like.
- suitable solvents such as haiogenated hydrocarbons, e.g., methylene chloride, ethylene chloride, dichloroethane and the like.
- DNA technology it is to be understood that the invention is not limited thereto but may be applied with equal facility to any biological system, and to any and all pharmaceutical products manufactured for parenteral use, i.e. anti-cancer drugs, anti-hypertension drugs, anti-lipodemia drugs, antidepressants, etc.
- Endotoxin was broken down into its components by incubation with 6N HCl for 10 minutes at 120oC.
- the esterification reaction was performed by first preparing diazomethane and then reacting diazomethane with the oligopeptide mixture.
- N-methyl-N-nitroso-N-nitroguanidine 99 mg were placed in the inner tube of a Pierce Microgenerator apparatus. Then 400 uL of H 2 O were added to the MNNG to form a suspension. Subsequently, 1500 uL of diethylether were dispensed in the bottom of the generator and the generator was placed in an ice bath. Using a 1 mL syringe fitted with a needle, 450 uL of 5N NaOH were then carefully injected into the inner tube containing MNNG. The generator then remained in the ice bath for 45 minutes and this reaction gave an approximately 60% yield of diazomethane.
- diazomethane required were estimated as follows: 99 mg of MNNG yielded approximately 400 ⁇ moles of diazomethane in 1500 uL and the number of moles of diazomethane required was approximately 100 times the number of moles of protein that was hydrolyzed.
- Dry MeOH to a final volume of 100 uL was pipetted into the PICOTAG chamber and vcrtexed. 40 uL of dry MeOH was then pipetted into each sample tube and vcrtexed. 400 uL of diazomethane solution was then pipetted into the PICOTAG chamber and vortexed. After calculating the amount of diazomethane required as described above, 10 times this amount was pipetted into each sample tube and then vortexed. Any remaining diazomethane solution was added to the PICOTAG chamber. The closed PICOTAG then stood at. room temperature for 4 hours. The excess reagent was removed from the PICOTAG chamber. The reagents from the sample tubes were then removed in the PICOTAG workstation under vacuum (30-60 mTorr).
- MeTFA Methyltrifiuoroacetate
- MeTFA Methyltrifiuoroacetate
- TEA triethylamine
- the reagent was removed in the PICOTAG workstation under vacuum (30-50 mTorr).
- 500 uL of MeTFA:MeOH was pipetted into the PICOTAG chamber and vortexed.
- 2 uL of TEA were added to each sample tube and vortexed.
- 6 uL of MeTFA:MeOK were added to each sample tube and vortexed.
- the PICOTAG chamber was then pressurized four times with N 2 in the PICOTAG workstation.
- the closed PICOTAG chamber then stood at room temperature in the dark overnight.
- the excess reagents were then removed from the PICOTAG chamber and the chamber was washed with MeOH.
- the reagents were removed from the sample tubes in the PICOTAG workstation under vacuum (30 mTorr).
- This hydrolysis reaction was performed in order to destroy the amine-boron bonds. This was done by using a mixture of HCl and MeOH.
- the HCl/MeOH (IN) was prepared by diluting and mixing a vial (1 mL) of 3N HCl with 2 mL of dry MeOH. lmL of IN HCl/MeOH was then pipetted into the PICOTAG chamber and vortexed. 40 uL of IN HCl/MeOH was then added to each sample tube and vortexed.
- the PICOTAG chamber was heated in the PICOTAG workstation at 97°C for 20. minutes. The excess reagent was removed from the PICOTAG chamber. The reagents were removed from the sample tubes in the PICOTAG workstation under vacuum (100 mTorr).
- the derivatized components of endotoxin were extracted with methylene chloride (CH 2 Cl 2 ) in order to prevent unwanted by-products from interfering with the gas chromatography and mass spectrometry.
- CH 2 Cl 2 methylene chloride
- 200 uL of CH 2 Cl 2 was added to each sample tube and each tube was vortexed individually for 3 minutes.
- the CH 2 Cl 2 was then decanted into pre-weighed pyrex tubes and saved (Batch I). This step removed by-products which were soluble in organic solvents (and the reduced derivative of beta-hydroxymyristic acid from the aqueous phase which contains the polyamino alcohol derivatives).
- a saturated solution of potassium carbonate (K 2 CO 3 ) was prepared and washed with CH 2 Cl 2 and then filtered. With the sample tubes in horizontal position, 10 uL of the K 2 CO 3 solution was then added to the top of each sample tube one at a time. The sample was then vortexed as the drop descended to the bottom of the tube. The sample tube was tilted so the drop moved to the top of the tube and the vortex procedure was then repeated. This procedure ensured that the sample and the K 2 CO 3 solution were properly mixed. 200 uL of CH 2 Cl 2 were then added to each sample tube, which were then vortexed for 1 minute, and the phases were allowed to separate. The eupernatent was then carefully decanted into preweighed pyrex tubes (Batch II). The supernatent then contained the polyamino alcohols. The aqueous portion which remained behind contained the by-products that were soluble only in water.
- the CH 2 Cl 2 was removed frcm the sample tubes (Batch I and Batch II) in the PICOTAG workstation under vacuum, which was 150 mTorr.
- the sample tubes were then weighed to calculate the amount of material present. (If the weight is too high, it is likely that one agueous phase containing the K 2 CO 3 has contaminated the sample. If this occurs, wash the sample with 200 uL cf CH 2 Cl 2 , decant and remove the CH 2 Cl 2 from the sample tubes under vacuum [150 mTorr]).
- the components of endotoxin were separated on a gas chromatograph and introduced into the ion source of the mass spectrometer.
- the derivatized sample (0.5 uL) was injected into an Ultra-1 capillary column (0.33 urn film thickness, 200 um internal diameter, 50 m length) via an on-column injector.
- the analysis was performed with a flow rate of 0.41 mL/minute under the following conditions: Initial temperature: 70°C,
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
L'invention concerne un procédé de détection d'endotoxine dans des compositions de polypeptide. En particulier, cette invention concerne un procédé de mesure de l'endotoxine par quantification d'un composant volatile dérivé de l'endotoxine, l'acide bêta-hydroxymyristique, présent dans les compositions de polypeptide. La présente invention peut être utilisée pour déterminer la teneur en endotoxine de produits destinés à être utilisés en médecine clinique, y compris les produits préparés par une technologie d'ADN de recombinaison.The invention relates to a method for detecting endotoxin in polypeptide compositions. In particular, this invention relates to a method of measuring endotoxin by quantifying a volatile component derived from endotoxin, beta-hydroxymyristic acid, present in the polypeptide compositions. The present invention can be used to determine the endotoxin content of products for use in clinical medicine, including products prepared by recombinant DNA technology.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US34748589A | 1989-05-04 | 1989-05-04 | |
US347485 | 1989-05-04 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0423312A1 true EP0423312A1 (en) | 1991-04-24 |
EP0423312A4 EP0423312A4 (en) | 1991-08-28 |
Family
ID=23363893
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19900907628 Withdrawn EP0423312A4 (en) | 1989-05-04 | 1990-05-03 | Detection of endotoxin in pharmaceutical preparations by mass spectrometry |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0423312A4 (en) |
JP (1) | JPH04501315A (en) |
AU (1) | AU5641990A (en) |
CA (1) | CA2032133A1 (en) |
WO (1) | WO1990013534A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014161613A1 (en) * | 2013-04-04 | 2014-10-09 | Universite De Bourgogne | A method for evaluating the level of neutralization of the biological activity of lipopolysaccharides (lps) in a sample |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4017077A (en) * | 1975-07-03 | 1977-04-12 | Bally Manufacturing Corporation | Matrix transformation pin ball machine with score multiplier option |
US4038029A (en) * | 1976-01-20 | 1977-07-26 | Worthington Biochemical Corporation | Limulus lysate turbidity test for pyrogens |
US4093381A (en) * | 1976-11-29 | 1978-06-06 | Karamian Narbik A | Method for assaying endotoxins |
-
1990
- 1990-05-03 CA CA002032133A patent/CA2032133A1/en not_active Abandoned
- 1990-05-03 EP EP19900907628 patent/EP0423312A4/en not_active Withdrawn
- 1990-05-03 JP JP50756990A patent/JPH04501315A/en active Pending
- 1990-05-03 AU AU56419/90A patent/AU5641990A/en not_active Abandoned
- 1990-05-03 WO PCT/US1990/002459 patent/WO1990013534A1/en not_active Application Discontinuation
Non-Patent Citations (2)
Title |
---|
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 75, no 8, August 1978, pages 3993-3997, Washington, DE, US; S.K. MAITRA et al. "Determination of lipid A and endotoxin in serum by mass spectroscopy" * |
See also references of WO9013534A1 * |
Also Published As
Publication number | Publication date |
---|---|
JPH04501315A (en) | 1992-03-05 |
EP0423312A4 (en) | 1991-08-28 |
WO1990013534A1 (en) | 1990-11-15 |
CA2032133A1 (en) | 1990-11-05 |
AU5641990A (en) | 1990-11-29 |
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Legal Events
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PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
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18W | Application withdrawn |
Withdrawal date: 19920709 |