WO1990013534A1

WO1990013534A1 - Detection of endotoxin in pharmaceutical preparations by mass spectrometry

Info

Publication number: WO1990013534A1
Application number: PCT/US1990/002459
Authority: WO
Inventors: J. Wayne Cowens; Jeannine A. Maide
Original assignee: Cowens J Wayne; Maide Jeannine A
Priority date: 1989-05-04
Filing date: 1990-05-03
Publication date: 1990-11-15
Also published as: JPH04501315A; EP0423312A1; EP0423312A4; CA2032133A1; AU5641990A

Abstract

The present invention relates to a method of detection of endotoxin in polypeptide compositions. In particular, this invention contemplates a method for measuring endotoxin by quantitating the amount of a volatile derivatized component of endotoxin, beta-hydroxymyristic acid, present in polypeptide compositions. The present invention can be used for testing the endotoxin content of products which are intended for use in clinical medicine, including those produced by recombinant DNA technology.

Description

DETECTION OF ENDOTOXIN IN PHARMACEUTICAL

PREPARATIONS BY MASS SPECTROMETRY

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 σuantitating 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. 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.

However, the central problem in marketing such products for use in clinical medicine is the development of quality control procedures for insuring the product's identity, purity and biological potency as required by the

FDA. One of the most critical quality control tests required by the FDA for all drugs is the measurement of endotoxin in the final formulation. This test is particularly important for drugs manufactured by recombinant DNA technology in microorganisms. The standard test for endotoxin measurement has been the limulus test, which is based on the ability of endotoxin to cause the lymph of the horseshoe crab to clot.

Although this bioassay is relatively sensitive, its main drawbacks, lie in the fact that it is not sufficiently specific, it is a difficult test to standardize and it is not easily reproducible.

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 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

alcohols from the reduced hydrolysate; and detecting beta-hydroxymyristyl alcohol by mass spectrometry.

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. The term endotoxin as used in the specification and claims also includes gram-negative bacterial pyrogen and lipopolysaccharide (LPS).

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 (-CH₂OH) 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:

a) hydrolysis of' endotoxin in the polypeptide composition into its component parts;

b) esterification of the resulting hydrolysate with an alkanol which forms, inter alia, an alkyl ester of beta-hydroxymyristic acid; c) acylation of amino groups present in the hydrolysate;

d) reduction of the carbalkoxy groups of the esters formed in the hydrolysate to CH₇OH;

e) selective extraction of the beta- hydroxymyristyl alcohol from the reduced hydrolysate;

f) 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. In such reduction, amino groups of the hydrolysate, if present, will complex with the boron of the reducing reagent.

It is usually preferred to hydrolyze the resulting reduction mixture to destroy the amine-boron bonds in order to solubilize the reduction mixture.

In the extraction step previously described, 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.

While the invention, as described has been found to be particularly useful in connection with measuring the endotoxin content of protein products produced by recombinant

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.

The following examples assist in further detailing the subject invention herein without in any way limiting same.

EXAMPLES

1. Hydrolysis of Endotoxin

Endotoxin was broken down into its components by incubation with 6N HCl for 10 minutes at 120ºC.

2. Derivatization Chemistry

A. Esterification

The esterification reaction was performed by first preparing diazomethane and then reacting diazomethane with the oligopeptide mixture.

a. Preparation of Diazomethane

99 mg of N-methyl-N-nitroso-N-nitroguanidine ( MNNG ) were placed in the inner tube of a Pierce Microgenerator apparatus. Then 400 uL of H₂O 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.

b. Reaction of Diazomethane with Endotoxin

Components

The amounts of diazomethane required were estimated as follows: 99 mg of MNNG yielded approximately 400 μmoles 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).

B. Acylation

Freshly distilled Methyltrifiuoroacetate (MeTFA) was prepared and then 500 uL of MeTFA was mixed with 500 uL of dry MeOH. 2 uL of triethylamine (TEA) was added to each sample tube and vortexed. 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 N₂ 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).

C. Reduction

Boron trideuteride (1M) in tetrahydrcfuran was used as the reducing reagent in this reaction. 800 uL of Boron trideuteride (B₂D₆) in tetrahydrofuran (THF) was pipetted into the PICOTAG chamber and vortexed. 100 uL of 1M B ₂D₆,/THF was then pipetted into each sample tube. The PICOTAG chamber was pressurized four times with N₂, and then heated in the PICOTAG workstation at 90°C for 30 minutes. The chamber was periodically removed and vcrtexed.

The excess reagent was removed from the chamber, which vfas then washed with dry MeOH. The chamber was vortexed before the MeOH was removed. Dry MeOH (20 uL) was then slowly added to each sample tube to decompose the reducing reagent. The reagents were then removed from the sample tubes in the PICOTAG workstation under vacuum (200 mTorr). After reduction was completed, all amine groups were compiexed to the Boron atom.

D. Hydrolysis of Boranes

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 above steps were then repeated as follows: 1N HCl/MeOH (1 mL) was added to the PICOTAG chamber and vortexed, IN HCl/MeOH (40 uL) was added to each sample tube and vortexed. The PICOTAG chamber was heated for 20 minutes at 97°C. The excess reagent was removed from the PICOTAG chamber. However, at this stage, the reagents from the sample tubes were removed under vacuum of 45 mTcrr.

E. Extraction

The derivatized components of endotoxin were extracted with methylene chloride (CH₂Cl₂) in order to prevent unwanted by-products from interfering with the gas chromatography and mass spectrometry. 200 uL of CH₂Cl₂ was added to each sample tube and each tube was vortexed individually for 3 minutes. The CH₂Cl₂ 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 (K₂CO₃) was prepared and washed with CH₂Cl₂ and then filtered. With the sample tubes in horizontal position, 10 uL of the K₂CO₃ 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 K₂CO₃ solution were properly mixed. 200 uL of CH₂Cl₂ 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 CH₂Cl₂ 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 K₂CO₃ has contaminated the sample. If this occurs, wash the sample with 200 uL cf CH₂Cl₂, decant and remove the CH₂Cl₂ from the sample tubes under vacuum [150 mTorr]).

F. Silylation

For each 500 ug of original sample, 4 uL of pyridine and 30 uL of TMSDEA (trimethylsilyl diethylamine) were added to samples frcm Batch I and Batch II; each sample was then vortexed for 1 minute. The PICOTAG cnamber was then heated at 56°C for 20 minutes. This reaction added a trimethylsilyl group to the free alcohol groups. The sample tubes were capped and stored at -20°C until assay.

3. Separation

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,

Initial time: 0, Rate (C/Min) = 1.5, Final temperature =

310°C, Final time = 5 minutes.

Heated Zones:

Oven (Standby) Setpoint: 70°C

Oven (Standby) Limit: 325°C

Injection Port B Setpoint: 70°C

Injection Port B Limit: 325°C

Ion Source: 350°C

Transfer Line Setpoint: 32C°C

Transfer Line Limit: 350°C

Under these ccnditicns, the derivatized alcohol of beta-hydroxymyristic acid eluted at 47.67 minutes (retention index of 1950). Ions with m/e 257 and 221 were not detected in the spectra of any other ccmpcund in the hydrolysate of endotoxin.

Claims

WE CLAIM:

1. A method for detection of endotoxin in a polypeptide composition comprising the steps of:

a) hydrolysis of the composition

containing said endotoxin, to form beta-hydroxymyristic acid;

b) esterifying the resulting hydrolysate

with an alkanol;

c) reducing the carbalkoxy groups present in the hydrolysate to hydroxymethyl groups;

d) separating the volatile alcohols from the reduced hydrolysate; and

e) detecting beta-hydroxymyristyl alcohol by mass spectrometry.

2. The method of Claim 1 wherein said polypeptide composition is produced by recombinant DNA technclogy.

3. A method for detection of endotoxin in a polypeptide composition comprising the steps:

a) hydrclysis of the polypeptide

composition;

b) esterification of the resulting

hydrolysate with an alkanol;

c) acylation of free amino groups present in the hydrolysate;

d) reduction of the carbalkoxy groups in the hydrolysate to hydroxymethyl groups;

e) extraction of beta-hydroxymyristyl

alcohol from the reduced hydrolysate; f) derivatizion of the extracted alcohol; g) separating the volatile derivatized

product from the hydrolysate, and

h) detecting and quantitating the volatile derivatized alcohol of beta-hydroxymyristic acid by mass spectrometry.

4. The method of Claim 3, wherein said reduction of step (d) is accomplished by a borohydride reducing agent.

5. The method of Claim 3, wherein said extraction of step (e) is performed with a halogenated hydrocarbon.

6. The miethod of Claim 5 wherein said eolvent is methylene chloride, ethylene chloride or dichloroethane.

7. The method of Claim 3, wherein said separation of step (g) is accomplished by gas chrcmatography or column chromatography.

8. The method of Claim 3, wherein said polypeptide compositions are pharmaceutical parental preparations.