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107

Chapter 6

“BACTERIAL ENDOTOXINS QUANTIFICATION

IN VETERINARY INJECTIONS,

INTERMEDIATES, OIL BASED INJECTIONS

AND OTHER DRUGS”

108

6.1 Veterinary Drugs:

Introduction:

Like humans, animals also respond physiologically to the Endotoxins in the

same fashions to various primary and secondary responses upon

administration of the injectables. Same Endotoxins limits are applicable to

veterinary as of humans, based on the body weight. The bacterial Endotoxins

test to be passed as per the respective monograph before releasing the drug

into market but due to interfering factors in most of the cases there is a

probability of getting false positive results. So Non-Interfering Dilution (NID) to

be selected before validating a product. Four products were validated and

the NID factor among those veterinary injections is between + two fold.

For most of the new veterinary formulations the Endotoxin limits are not

available in pharmacopeia80, In that case the Endotoxin limit of such drugs

need to be calculated based on the body weight of the animal. It is prudent

to check clinical publications to determine if there are dosage patterns that

exceed the labeled dose range.

For example to new formulation name “X” with potency 500mg/mL.

Endotoxin limits was not available in pharmacopeia, wishing to analyze. The

“X” can be prescribed to hen, dog and buffalo. Lets quote an example, how

to calculate Endotoxin Limit to a new drug based on the body weight of the

animal.

Hen: Maximum allowable dosage per hour is 0.25mL (125mg).

Body weight is 1.5 Kg.

Dog: Maximum allowable dosage per hour is 2 mL (1000mg).

Body weight is 15 Kg.

Buffalo: Maximum allowable dosage per hour is 10 mL (2500 mg).

Body weight is 300 Kg.

The K/M formula

The K/M formula was presented in the FDA LAL-test Guideline (1987) as a way

to calculate the Endotoxin limit, as shown below.

Endotoxin Limit (EL) = K (tolerance limit)

M (maximum dose/kg) (hr.)

6.1 Veterinary Drugs

109

For Hen:



= K 5Eu/Kg/hr .

125 mg/1.5/kg) (hr.)

=0.06 Eu/mg

For Dog:



= K 5Eu/Kg/hr .

1000 mg/15/kg) (hr.)

=0.075 Eu/mg

For Buffalo:



= K 5Eu/Kg/hr .

2500 mg/300/kg) (hr.)

=0.6 Eu/mg

Based on the body weight and the dosage of the drug the Endotoxin limit

varies. Lesser the body weight the limits are more stringent.

Materials and Methods:

Materials:

Lyophilized Limulus Amoebocyte Lysate of 0.125 sensitivity (LAL), Control

Standard Endotoxin 5 Eu/ng (CSE), LAL Reagent Water (LRW) of Endosafe US,

Depyrogenated (250°C for 30 min )10 X 75 mm assay tubes, 16X100 mm

dilution tubes ,pyrogen free Micropipette tips, vortex mixture, 1N NaoH, 1N

HCL, Meloxicam, Dexamethasone phosphate and Ivermectin were used for

determination of Endotoxin content by the gel clot technique.

The sensitivity of the Lysate (labeled 0.125 Eu/mL) was determined by using

known amount of E.coli Control Standard Endotoxin.


110

In the gel-clot techniques, the reaction end point is determined from dilutions

of the material under test in direct comparison with parallel dilutions or a

reference Endotoxin, and quantities of Endotoxins are expressed in Endotoxin

units69.

1. Preparation of Standard stock solution and standard solutions: The CSE

having a defined potency of 50 EU/Vial was reconstituted with 5ml of LRW

and mixed intermittently for 30 minutes using a vortex mixture and this

concentrate was used to prepare 2λ, λ, λ/2 & λ/4, where λ is the labeled

claim sensitivity of Lysate.

2. Preparation of sample solution: Test samples were diluted to the required

concentrations based on the formulae MVD. MVD is the maximum valid

dilution, which is allowable dilution of the specimen at which the Endotoxin

limit can be determined. The general equation to determine MVD is

MVD = (Endotoxin limit X Concentration of sample solution)/ (λ). Where E.L is

the Endotoxin limit of the test sample, which is specified in the individual

monograph in terms of volume or units of active drug (in EU/mg).

3. Meloxicam sample preparation: Potency= 5mg/mL , E.L= 10 Eu/mg , Lysate

sensitivity is 0.125 Eu/mL and MVD = 400.The following test dilutions are

prepared by 1:400 (0.01 mg/mL), 1:200 (0.03 mg/mL), 1:100 (0.05 mg/mL), 1:50

(0.10 mg/mL) & 1:25 (0.20 mg/mL).

4. Dexamethasone Phosphate sample preparation: Potency= 3.34 mg/mL,

E.L= 31.30 Eu/mg, Lysate sensitivity is 0.125 Eu/mL and MVD = 836. The following

test dilutions are prepared by 1:836 (0.004 mg/mL), 1:418 (0.008 mg/mL), 1:209

(0.016 mg/mL), 1:104 (0.033 mg/mL) & 1:52 (0.066 mg/mL).

5. Ivermectin sample preparation: Potency= 10 mg/mL, E.L= 5 Eu/mg, Lysate

sensitivity is 0.125 Eu/mL and MVD = 400. The following test dilutions are

prepared by 1:400 (0.025 mg/mL), 1:200 (0.05 mg/mL), 1:100 (0.10 mg/mL),

1:50 (0.20 mg/mL) & 1:25 (0.40 mg/mL).


111

Methods

Equal volume of test sample and LAL reagent is added in a

depyrogenated test tube of 10 X 75 mm and incubate this mixture at 37± 1°C

for 60±2 min. Then invert the tube by 180° and look for gel formation. If a gel

inside the test tube is able to maintain its integrity after inverting the tube to

180° then it is a positive reaction which indicates presence of Endotoxin in the

sample greater than the limit. Other than this any condition is considered as

negative which indicates absence of Endotoxin in the sample (lesser than the

lysate sensitivity).

Product Testing: For testing products equal volume of drug (sample) and LAL

reagent is taken and following tubes are prepared)

Negative Product Control (NPC) - Sample + LAL

Positive Product Control (PPC) - Sample + CSE (2λ) + LAL

Negative Water Control (NWC) - LRW + LAL

Positive Water Control (PWC) - LRW + CSE (2λ) + LAL

Majority of times it has been a common observation that if a product is tested

directly it inhibits the LAL test and thus shows interference

Interference: Interference is defined as a significant difference between the

end points of positive water control and positive product control using

standard Endotoxin.

This interference could be either inhibition wherein the recovery of Endotoxin is

below than the expected or enhancement wherein the recovery of Endotoxin

is higher than expected.

Product Validation: Product needs to be validated before start for routine

testing. Validation is a test condition where an Endotoxin standard is detected

with the same efficiency in a test sample as it is in LRW. This validation study

consists of two different phases wherein in Phase I (Preliminary screening)

involve interference testing and Phase II consists of validation of product.

Significance of product validation is that it gives information on

whether there are any interfering factors in the drug product to the LAL test

and also it gives an idea of the approximate levels of Endotoxin content in the

drug product. It also covers manufacturing of product and formulation of

the product.


112

It is always advisable to carry out revalidation if product formulation is

changed and which is likely to affect the interference pattern of the product

for LAL test. Also revalidation is to be conducted for any product if there is any

change in manufacturing procedures or in vendor.

6.1 A: Meloxicam Preliminary Screening Test:

Product : Meloxicam

Concentration : 5mg/ml

Endotoxin Limit : 10 EU/mg

MVD : 400

Test Dilution : 1:25, 1:50, 1:100, 1:200 and 1: 400

Test Concentrations : 0.20mg/mL, 0.10mg/mL, 0.05mg/mL,

0.03mg/mL and 0.01mg/mL

Phase I: Preliminary Screening / interference Study25 (Cooper, 1990).

In this two identical series of product dilutions (two-fold dilutions), one

spiked with 2λ, and one left unspiked. The result of Phase I will tell you the

Non-Interfering Dilution (NID) of the product. The Non-Interfering Dilution (NID)

is the first set of PPC that shows a gel.

Test Dilutions /

Concentrations

NPC PPC

1:25 − − + +

1:50 − − + +

1:100 − − + +

1:200 − − + +

1:400 − − + +

++: Gel Formation − − : No Gel Formation

Conclusion: pH of the product was adjusted to 7-8 at initial dilution, carried

out screening and observed Non Interfering Dilution (NID) at 1/16

MVD (1:25). Non Interfering Concentration is 0.20mg/ mL.

Endotoxin content is < 10EU/ mg.


113

End-Product Endotoxins Test:

Product : Meloxicam

Preparation : Product Concentration : 5 mg / mL


MVD : 400

Test Dilution : 1:50

Test Concentration : 0.10 mg/mL

Results :

Test Endotoxin

Concentration EU/mL.

NEG

Control

NEG

Product

Control

Test

End

point.

Log of

End

point.

Geometric Mean

=A log (log of End

point.)

Material 2λ λ ½λ ¼λ EU/mL

+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − 0.125 -0.9030

CSE

Water

Control

+ + - − 0.125 -0.9030

+ + + − − 0.0625 -1.2041

+ + + − − 0.0625 -1.2041

+ + - − − 0.125 -0.9030

Positive

Product

Control

+ + - − − 0.125 -0.9030

Antilog

(-3.612/4)

=Antilog –0.903

=0.125EU/mL

Antilog

(-4.2142/4)

=Antilog–1.05355

=0.088EU/mL

Interpretation :

Test results are : Valid

Comments : Product validation for Meloxicam was carried out at MVD/8

(1:50) (Test Concentration: 0.10mg/mL) after adjusting the pH81.

The GM of the end points in Endotoxin / LRW was 0.125EU/mL

and Endotoxin/product was 0.088 EU/mL which is in between 2λ

and ½ λ, which indicates no inhibition and enhancement at test

dilution MVD / 8 (1:50).


114

6.1 B: Dexamethasone Phosphate Preliminary Screening Test:

Product : Dexamethasone Phosphate

Concentration : 3.34 mg/ml

Endotoxin Limit : 31.30 EU/mg

MVD : 836

Test Dilution : 1:52, 1:104, 1:209, 1:418 and 1:836

Test Concentrations : 0.066mg/mL, 0.033mg/mL, 0.017mg/mL,

0.008mg/mL and 0.004mg/mL.

In this two identical series of product dilutions (two-fold dilutions), one spiked

with 2λ, and one left unspiked. The result of Phase I will tell you the Non-

Interfering Dilution (NID) of the product. The Non-Interfering Dilution (NID) is the

first set of PPC that shows a gel.

Test Dilutions /

Concentrations

NPC PPC

1:52 (0.066 mg/mL) + + + +

1:104 (0.033 mg/mL) − − + +

1:209 (0.016 mg/mL) − − + +

1:418 (0.008 mg/mL) − − + +

1:836 (0.004 mg/mL) − − + +





Endotoxin content is < 31.30EU/ mg.


115


Product : Dexamethasone Phosphate

Preparation : Product Concentration : 3.34 mg / mL

Endotoxin Limit : 31.30 EU/mg

MVD : 836



Results :

Test Endotoxin


NEG

Control

NEG

Product

Control

Test

End

point.

Log of

End

point.

Geometric Mean

=A log (log of End

point.)


+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − 0.125 -0.9030

CSE

Water

Control

+ + - − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + + − − 0.0625 -1.2041

+ + - − − 0.125 -0.9030

Positive

Product

Control

+ + + − − 0.0625 -1.2041

Antilog

(-3.612/4)

=Antilog –0.903

=0.125EU/mL

Antilog

(-4.2142/4)

=Antilog–1.05355

=0.088EU/mL

Interpretation :


Comments : Product validation for Dexamethasone Phosphate was carried

out at MVD/4 (1:209) (Test Concentration: 0.016mg/mL) after

adjusting the pH. The GM of the end points in Endotoxin / LRW

was 0.125EU/mL and Endotoxin/product was 0.088 EU/mL which

is in between 2λ and ½ λ, which indicates no inhibition and

enhancement at test dilution MVD/4 (1:209).


116

6.1 C: Ivermectin Preliminary Screening Test:

Product : Ivermectin

Concentration : 10 mg/ml


MVD : 400

Test Dilution : 1:25, 1:50, 1:100, 1:200 and 1:400

Test Concentrations 0.40mg/mL, 0.20mg/mL, 0.10mg/mL, 0.05mg/mL

and 0.025mg/mL.



Interfering Dilution (NID) of the product. The Non-Interfering Dilution (NID) is the

first set of PPC that shows a gel.

Test Dilutions /

Concentrations

NPC PPC

1:12 (0.83 mg/mL) − − + +

1:25 (0.40 mg/mL) − − + +

1:50 (0.20 mg/mL) − − + +

1:100 (0.10 mg/mL) − − + +

1:200 (0.05 mg/mL) − − + +





Endotoxin content is < 5EU/ mg.


117


Product : Ivermectin

Preparation : Product Concentration : 10 mg / mL

Endotoxin Limit : 5EU/mg

MVD : 400



Results :

Test Endotoxin


NEG

Control

NEG

Product

Control

Test

End

point.

Log of

End

point.

Geometric Mean

=A log (log of End

point.)


+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − 0.125 -0.9030

CSE

Water

Control

+ + - − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

Positive

Product

Control

+ + - − − 0.125 -0.9030

Antilog

(-3.612/4)

=Antilog –0.903

=0.125EU/mL

Antilog

(-3.612/4)

=Antilog –0.903

=0.125EU/mL

Interpretation :


Comments : Product validation for Ivermectin was carried out at MVD/8

(1:50) (Test Concentration: 0.20mg/mL) after adjusting the pH.

The GM of the end points in Endotoxin / LRW was 0.125EU/mL

and Endotoxin/product was 0.125 EU/mL which is in between 2λ

and ½ λ, which indicates no inhibition and enhancement at test

dilution MVD/8 (1:50).

118

6.2 Intermediates

The schematic drug manufacturing process initiate with the starting raw

material proceeded with intermediated/stages and ends with the final

formulation. In order to comply the Endotoxin limit of the final drug product

the level of the Endotoxins in the raw materials used or intermediates formed

to be controlled so that the Endotoxin content in the final drug product was

controlled. It is always advisable to check intermediates and key starting raw

materials for Endotoxins and controlled at lower levels than the final drug

product in which they are used. So that the final drug product will pass the

bacterial Endotoxins test.


Materials:





HCL and Sodium Bicarbonate. were used for determination of Endotoxin

content by the gel clot technique.






units.










6.2 Intermediates

119




3. Sodium Bicarbonate sample preparation: Potency = 0.5952 mEq/mL, E.L= 5

mEq/mL , Lysate sensitivity is 0.125 Eu/mL and MVD = 20 .The following test

dilutions are prepared by 1:20, 1:10, 1:5 & 1:2.5).

Methods

Equal volume of test sample and LAL reagent is added in a

depyrogenated test tube of 10 X 75 mm and incubate this mixture at 37± 1°C

for 60±2 min. Then invert the tube by 180° and look for gel formation. If a gel

inside the test tube is able to maintain its integrity after inverting the tube to

180° then it is a positive reaction which indicates presence of Endotoxin in the

sample greater than the limit. Other than this any condition is considered as

negative which indicates absence of Endotoxin in the sample (lesser than the

lysate sensitivity).

Product Testing: For testing products equal volume of drug (sample) and LAL

reagent is taken and following tubes are prepared)

Negative Product Control (NPC) - Sample + LAL

Positive Product Control (PPC) - Sample + CSE (2λ) + LAL

Negative Water Control (NWC) - LRW + LAL

Positive Water Control (PWC) - LRW + CSE (2λ) + LAL

6.2 Intermediates

120

6.2 A: Sodium Bicarbonate Preliminary Screening Test:

Product : Sodium Bicarbonate

Concentration : 0.5952 mEq/mL

Endotoxin Limit : 5 EU/mEq

Molecular weight of Sodium Bicarbonate (NaHCO3) = 23 + 1 +12 + 48 = 84

1 Molar = 84 gms in 1000 mL = 84 mg/mL

1 millimole = 0.084 mg/mL

1 gm equivalent = Molarity / no. of replaceable H ions per liter.

= 84 mg/mL

1 / 1000 mL

1 milli equivalent = milli Molarity / no. of replaceable H ions per liter

= 0.084 mg/mL

1 / 1000 mL

= 0.084 mg x 1000

= 84mg /mL

Potency of Product = 50 mg/mL

= (50/84) mEq/mL

= 0.5952 mEq/mL

MVD = Potency of a product x Endotoxin limit / Lysate sensitivity

= 0.5952 mEq/mL x 5 EU/mEq

0.125 EU/mL

= 23.8

MVD : 23.8

It is recommended to take MVD as 20.

6.2 Intermediates

121

MVD = 20

MVD/2 =10

MVD/4 = 5

MVD/8 = 2.5

Test Dilutions: 1:2.5, 1:5, 1:10 and 1: 20

Test Dilutions / Concentrations NPC PPC

1:2.5 (0.0625 mEq/mL) − − + +

1:5 (0.1250 mEq/mL) − − + +

1:10 (0.2501 mEq/mL) − − + +

1:20 (0.5002 mEq/mL) − − + +


Conclusion: Carried out screening and observed Non Interfering Dilution

(NID) at 1/8 MVD (1:2.5). Non Interfering Concentration is (0.0625

mEq/mL). Endotoxins content is < 5u/ mEq.

6.2 Intermediates

122

End-Product Endotoxins Test79:

Product : Sodium Bicarbonate

Preparation : Product Concentration : 0.5952 mEq/mL

Endotoxin Limit : 5 EU/mEq

MVD : 23.8

Test Dilution : 1:5

Test Concentration : 0.1250 mEq/mL

Results : Test Endotoxin


NEG

Control

NEG

Product

Control

Test

End

point.

Log of

End

point.

Geometric Mean

=A log (log of End

point.)


+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − 0.125 -0.9030

CSE

Water

Control

+ + - − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

Positive

Product

Control

+ + - − − 0.125 -0.9030

Antilog

(-3.612/4)

=Antilog –0.903

=0.125EU/mL

Antilog

-3.612/4)

=Antilog –0.903

=0.125EU/mL

Interpretation :


Comments : Product validation for Sodium Bicarbonate was carried out at

¼ MVD (1:5) (Test Concentration: 0.1250 mEq/mL) after adjusting the pH. The

GM of the end points in Endotoxin / LRW was 0.125EU/mL and

Endotoxin/product was 0.125 EU/mL which is in between 2λ and ½ λ, which

indicates no inhibition and enhancement at test dilution MVD / 4 (1:5).

123

6.3 Oil Based Injections:


The prototypic examples of Endotoxins are lipopolysaccharides (LPS) or Lipo-

oligo-Saccharides (LOS) found in the outer membrane of various gram-

Negative bacteria and are an important cause of their ability to cause fever,

a lowering of blood pressure and activation of inflammation and

coagulation. Endotoxins are the large part responsible for the dramatic

clinical manifestations of infections with Pathogenic gram negative bacteria.

In pharmaceutical production, it is necessary to remove all traces of

Endotoxins from drug product containers as even small amounts of Endotoxins

will cause illness in humans.

A very sensitive assay for detecting presence of Endotoxins is the Limulus

Amoebocyte Lysate assay, utilizing blood from Horseshoe crab. Very low

amounts of LPS can cause coagulation of lysate due to a powerful

amplification through Enzymatic cascade.

Even though Lysate is very sensitive in Endotoxins detection but in case of

Hormones and Oil based products, we have observed false negative results

due to non availability of the Endotoxins to the Lysate. It was observed that

vigorous vortexing of the test sample in LRW with valid dilution will disperse the

Endotoxins into the aqueous media and thus available to lysate. We have

cross checked the technique by spiking known concentration of Endotoxins

into the sample and achieved 100% recovery upon analysis after vigorous

vortexing.

Lyophilized Limulus Amoebocyte Lysate of 0.0625 & 0.0312 sensitivity

(LAL), Control Standard Endotoxin 5 Eu/ng (CSE), LAL Reagent Water (LRW) of

Endosafe US, Depyrogenated (250°C for 30 min) 10 X 75 mm assay tubes,

16X100 mm dilution tubes, pyrogen free Micropipette tips, vortex mixture, 1N

NaoH, 1N Hcl, Calcium gluconate Injection & Amosil (Silicon Oil) were used for

determination of NID by gel clot technique.

The sensitivity of the Lysate (labeled 0.0625 & 0.0312) was determined

by using known amount of E.coli Control Standard Endotoxin.

6.3 Oil Based Injections

124




units.










MVD = (Endotoxin limit X Concentration of sample solution)/ (λ).

Where E.L is the Endotoxin limit of the test sample, which is specified in the

individual monograph in terms of volume or units of active drug (in EU/mg).

3. Calcium gluconate Injection sample preparation: Potency=0.85 mg/ml,

E.L=0.17 EU/m, Lysate sensitivity is 0.625 Eu/mL and MVD = 2. The following test

dilutions are prepared by 1:1 (0.85 mg/ml) & 1:2 (0.42mg/mL).

4. Amosil (Silicon Oil) sample preparation: Potency=1 ml/ml, E.L=0.5 EU/ml,

Lysate sensitivity is 0.312 Eu/mL and MVD = 16. The following test dilutions are

prepared by 1:16, 1:8, 1:4 & 1:2.

Results and discussions:

Phase I: Preliminary Screening / interference Study25



Interfering Dilution (NID) of the product. The Non-Interfering Dilution (NID) is

the first set of PPC that shows a gel.

6.3 Oil Based Injections

125

Calcium gluconate Injection: Inhibition at 1:1

Sample Dilution 1:1 1:2

Unspiked -- --

Spiked -- ++

Table:1 In the above assay is showing interference (Inhibition) at 1:1 dilution even in the spiked samples with known concentration of controlled standard Endotoxin. Amosil (Silicon Oil): Inhibition at 1:2

Sample Dilution 1:2 1:4 1:8 1:16

Unspiked -- ++ -- --

Spiked -- ++ ++ ++

Table:2 In the above assay is showing interference (Inhibition) at 1:2 dilution even in the spiked samples with known concentration of controlled standard Endotoxin. Calcium gluconate Injection: NID-1:1(1:2 selected for validation)

Sample Dilution 1:1 1:2

Unspiked -- --

Spiked ++ ++

Table:3 The sample was vorted vigoursly for 20 min, after vortexing the product is showing NID at 1:1 dilution. Amosil (Silicon Oil): NID-1:8 (1:16 selected for validation)

Sample Dilution 1:2 1:4 1:8 1:16

Unspiked ++ ++ -- --

Spiked ++ ++ ++ ++

Table:4 The sample was vorted vigoursly for 20 min, after vortexing the product is

showing NID at 1:8 dilution.

This assay shows no inhibition from 1:1dilution onwards in Calcium gluconate

Injection and 1:8 Amosil (Silicon Oil) and the spike recovery from the NID

onwards. It is advisable to validate the product next to MVD to take care of

any batch to batch variation during regular production in the

pharmaceutical industries.

6.4 Other Drug Molecules

126

Phase II: Validation of Product

For validation, test and compare two identical series of Endotoxin dilutions

bracketingλ; One prepared in LRW and another prepared in product diluted

to the proposed test dilution. Here dilution selected for validation is refer table

1. (Hot spike method).

Phase: II Results:

Calcium gluconate Injection Amosil (Silicon Oil)

Repl

icat

es

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

0156

Eu

/mL

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

0156

Eu

/mL

0.

0078

Eu

/mL

1 + + - - + + - - 2 + + - - + + - - 3 + + - - + + - - 4 + + - - + + - -

Table: 5 Endotoxin/product; Negative product control: --; Geometric Mean = 0.0625

EU/ml for Calcium gluconate Injection & 0.0312 Eu/ml for Amosil (Silicon Oil).

Assay results of the products after spiking with known concentration of

Endotoxin are presented in Table 5.

Replicates 0.125 Eu/mL 0.0625 Eu/mL 0.0312 Eu/mL 0.0156 Eu/mL 1 + + - - 2 + + - - 3 + + - - 4 + + - -

Table: 6 Endotoxin/LRW; Negative product control: --; Geometric Mean = 0.0625 EU/ml. Assay results of label claim sensitivity of the lysate are presented in Table 3.

Replicates 0.0625 Eu/mL 0.0312 Eu/mL 0.0156 Eu/mL 0.0078 Eu/mL 1 + + - - 2 + + - - 3 + + - - 4 + + - -

Table: 7 Endotoxin/LRW; Negative product control: --; Geometric Mean = 0.0312 EU/ml.

Assay results of label claim sensitivity of the lysate are presented in Table 7.

Successful validation requires that both series confirm label claim (Geometric

mean) within +/- one two-fold dilution.

127

6.4 Other Drug Molecules:

Introduction:

During the research along with various drug substances, Intermediates, Drug

products and medical devices, we have studied the behavior of the other

drug products like anti-cancer drugs e.t.c in this chapter. The bacterial

Endotoxins test to be passed as per the respective monograph before

releasing these drugs into market but due to interfering factors in most of the

cases there is a probability of getting false positive results.


Materials:





HCL, Gentamycin sulphate, Rantidine Hcl, Ringer lactate, Ondansetron Hcl

Injection, Insulin, Insugen N, Diclofenac sodium, Aristoneurol Injection and

CB-12 Injection were used for determination of Endotoxin content by the gel

clot technique.






units.










128




3.Gentamycin Sulphate sample preparation: Batch No: GMS-0708,

Potency=80mg/2mL , E.L=1.67 Eu/mg, Lysate sensitivity is 0.125 Eu/mL and

MVD = 520. The following test dilutions are prepared by 1:520 (0.07 mg/mL),

1:260 (0.15 mg /mL), 1:130 (0.30 mg/mL), 1:65 (0.61 mg /mL) & 1:32 (1.25 mg

/mL).

4.Rantidine Hcl sample preparation: Batch No: RNH-0809, Potency=27.9

mg/mL , E.L= 7 Eu/mg , Lysate sensitivity is 0.125 Eu/mL and MVD = 1562. The

following test dilutions are prepared by 1:1560 (0.02 mg/mL), 1:780 (0.04

mg/mL), 1:390 (0.07 mg/mL) 1:195 (0.14 mg/mL) & 1:97 (0.29 mg/mL).

5.Ringer Lactate sample preparation: Batch No: RRL-0908, Potency= 1 mL/mL,

E.L=0.5 Eu/mL , Lysate sensitivity is 0.125 Eu/mL and MVD = 4. The following test

dilutions are prepared by 1:4, 1:2 & 1:1

6. Ondansetron Hcl Inj : Batch No: ONH-1008, Potency= 2 mg/mL, E.L=9.9

Eu/mg , Lysate sensitivity is 0.125 Eu/mL and MVD = 158. The following test

dilutions are prepared by 1:152 (0.013 mg/mL), 1:76 (0.026mg/mL), 1:38

(0.052mg/mL), 1:19 (0.105mg/mL) & 1:9.5 (0.210mg/mL).

7. Insulin sample preparation: Batch No: ISL-1108, Potency= 40 IU/ mL, E.L=

80Eu/100 = 0.8 Eu/IU, Lysate sensitivity is 0.125 Eu/mL and MVD = 256. The

following test dilutions are prepared by 1:256 (0.15 IU/mL), 1:128 (0.31 IU/mL),

1:64 (0.62 IU/mL), 1:32 (1.25 IU/mL) & 1:16 (2.5 mg/mL).

8. Insugen N (NPH) sample preparation: Batch No: ISN-1108, Potency= 40 IU/

mL, E.L= 80Eu/100 = 0.8 Eu/IU, Lysate sensitivity is 0.125 Eu/mL and MVD = 256.

The following test dilutions are prepared by 1:256 (0.15 IU/mL), 1:128 (0.31

IU/mL), 1:64 (0.62 IU/mL), 1:32 (1.25 IU/mL) & 1:16 (2.5 mg/mL).


129

9. Diclofenac Sodium sample preparation: Batch No: DCS-0109, Potency=25

mg/ mL, E.L= 4.60 Eu/mg , Lysate sensitivity is 0.125 Eu/mL and MVD = 920. The

following test dilutions are prepared by 1:920 (0.02 mg/mL), 1:460 (0.05

mg/mL), 1:230 (0.10 mg/mL), 1:115 (0.21 mg/mL) & 1:57 (0.44 mg/mL).

10. Aristoneurol Injection (Vitamin B1-B6-B12 Injection) sample preparation:

Batch No: ANI-0209, Potency= 1mL/ mL, E.L=116 Eu/mg , Lysate sensitivity is

0.125 Eu/mL and MVD = 928. The following test dilutions are prepared by

1:928, 1:464, 1:232, 1:116 & 1:58

11. CB - 12 Injection (CB-12 Injection consists of Ascorbic acid (Vitamin C))

sample preparation: Batch No: CBI-0209, Potency= 150 mg/ 1.5mL, E.L=1.2

Eu/mg, Lysate sensitivity is 0.125 Eu/mL and MVD = 960. The following test

dilutions are prepared by 1:960 (0.10 mg/mL), 1:480 (0.20 mg/mL), 1:240 (0.41

mg/mL), 1:120 (0.83 mg/mL) & 1:60 (1.66 mg/mL).

Results and discussions:

Gentamycin Sulphate: NID-1:32 (1:65 selected for validation)

Sample Dilution 1:32 1:65 1:130 1:260 1:520

Unspiked -- -- -- -- --

Spiked ++ ++ ++ ++ ++

Rantidine Hcl: NID-1:97 (1:195 selected for validation)

Sample Dilution 1:97 1:195 1:390 1:780 1:1560

Unspiked -- -- -- -- --

Spiked ++ ++ ++ ++ ++

Ringer Lactate NID-1:1 (1:2 selected for validation)

Sample Dilution 1:1 1:2 1:4

Unspiked -- -- --

Spiked ++ ++ ++


130

Ondansetron Hcl Injection : NID-1:9.5 (1:19 selected for validation)

Sample Dilution 1:9.5 1:19 1:38 1:76 1:152

Unspiked -- -- -- -- --

Spiked ++ ++ ++ ++ ++

Insulin: NID-1:32 (1:64 selected for validation)

Sample Dilution 1:16 1:32 1:64 1:128 1:256

Unspiked ++ -- -- -- --

Spiked ++ ++ ++ ++ ++

Insugen N (NPH): NID-1:32 (1:64 selected for validation)

Sample Dilution 1:16 1:32 1:64 1:128 1:256

Unspiked ++ -- -- -- --

Spiked ++ ++ ++ ++ ++

Diclofenac Sodium: NID-1:57(1:115 selected for validation)

Sample Dilution 1:57 1:115 1:230 1:460 1:920

Unspiked -- -- -- -- --

Spiked ++ ++ ++ ++ ++

Aristoneurol Injection: Vitamin B1-B6-B12 Injection)

NID-1:58 (1:116 selected for validation)

Sample Dilution 1:58 1:116 1:232 1:464 1:928

Unspiked -- -- -- -- --

Spiked ++ ++ ++ ++ ++

CB - 12 Injection: NID-1:240 (1:480 selected for validation)

Sample Dilution 1:60 1:120 1:240 1:480 1:960

Unspiked ++ ++ -- -- --

Spiked ++ ++ ++ ++ ++

Table: 1

This assay shows no inhibition from 1:32 in Gentamycin Sulphate, 1:97 in

Rantidine Hcl, 1:1 in Ringer Lactate,1:9.5 in Ondansetron Hcl Injection, 1:32 in

Insulin, 1:32 in Insugen N, 1:57 in Diclofenac Sodium, 1:58 in Aristoneurol


131

Injection and 1:240 in CB-12 Injection and the spike recovery from the NID

onwards. It is advisable to validate the product next to MVD to take care of

any batch to batch variation during regular production in the

pharmaceutical industries.

Phase II: Validation of Product

For validation, test and compare two identical series of Endotoxin dilutions

bracketingλ; One prepared in LRW and another prepared in product diluted

to the proposed test dilution. Here dilution selected for validation is refer table

1. (Hot spike method).

Phase: II Results:

Gentamycin Sulphate Rantidine Hcl

Repl

icat

es

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

1 + + - - + + - -

2 + + - - + + - -

3 + + - - + + - -

4 + + - - + + - -

Ringer Lactate Ondansetron Hcl Injection

Repl

icat

es

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

1 + + - - + + - -

2 + + - - + + - -

3 + + - - + + - -

4 + + - - + + - -


132

Insulin & Insugen N (NPH) Diclofenac Sodium

Repl

icat

es

0.25

Eu

/mL

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

1 + + - - + + - -

2 + + - - + + - -

3 + + - - + + - -

4 + + - - + + - -

Aristoneurol Injection

(Vitamin B1-B6-B12 Injection) CB - 12 Injection

Repl

ica

tes

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

Eu

/mL

0.

0312

Eu

/mL

0.

25

Eu/m

L

0.

125

Eu/m

L

0.

0625

u/

mL

0.

0312

Eu

/mL

1 + + - - + + - -

2 + + - - + + - -

3 + + - - + + - -

4 + + - - + + - -

Table: 2 Endotoxin/product; Negative product control: --; Geometric Mean =

0.125 EU/ml.

Assay results of the products after spiking with known concentration of

Endotoxin are presented in Table 2.

Replicates 0.25 Eu/mL 0.125 Eu/mL 0.0625 Eu/mL 0.0312 Eu/mL

1 + + - -

2 + + - -

3 + + - -

4 + + - -

Table: 3 Endotoxin/LRW; Negative product control: --; Geometric Mean = 0.125

EU/ml.

Assay results of label claim sensitivity of the lysate are presented in Table 3.

Successful validation requires that both series confirm label claim (Geometric

mean) within +/- one two-fold dilution. Validation is conducted at this dilution

on three batches of product.

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