Stages of production of escozine for pets
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Escozine for Pets™ has 4 major production steps. 1. Collection of Scorpions from the Scorpion Reservation. 2. Extraction of venom, purification and therapeutic dose preparation. 3. Polarization of extract and quality control of Polarization 4. Manufacturing, quality control, warehouse and shipment.

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Stages of Production of Escozine For Pets™

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Stages of production of escozine for pets

PETLIFE- White Paper

Stages of Production of Escozine For Pets™

Table of Contents

I.

Collection of Scorpions from the Scorpion Reservation. ..................................................... 2

II.

Extraction of Venom, Purification and Therapeutic Dose Preparation ............................. 4

III.Polarization of Extract and Quality Control of Polarization.................................................. 9

IV.Manufacturing, Quality Control, Warehouse And Shipment .............................................. 12

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Stages of production of escozine for pets

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Production Procedures

Escozine for Pets™ has 4 major production steps.

1. Collection of Scorpions from the Scorpion Reservation.

2. Extraction of venom, purification and therapeutic dose preparation.

3. Polarization of extract and quality control of Polarization

4. Manufacturing, quality control, warehouse and shipment.

I. Collection of Scorpions from the Scorpion Reservation

Dominican blue scorpions are from the Buthidae family: Rhopalurus Princeps.

They are collected from Medolife Corp’s 50,000m2 Scorpion Reservation, located in

the South of the Dominican Republic.

The reservation is divided in to SECTORS with numeric identification. Scorpions are

carefully collected every 22 days for venom extraction. These SECTORS are kept

separated from one another, but all scorpion populations are given the same care and

attention by Medolife veterinarians and biologists. The consistency in diet, breeding,

timing of extraction and population control is intended to support consistency of venom

concentration per each extraction.

Adult and young adult scorpions are chosen for the derived peptide extraction.

Scorpions collected for venom extraction are placed in small individual plastic

containers with individual numeric identification that specify the SECTOR they inhabit.

The plastic containers have holes on the top so that the scorpions have enough oxygen

during delivery to laboratory for extraction.

This numeric identification matches a collection calendar and thus prevents multiple

extractions of venom from same scorpions within a 22-day interval.

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II.

Extraction of Venom, Purification and Therapeutic Dose Preparation

Scorpions are collected for extraction in individual plastic containers and then delivered to the

laboratory where the pre-production process begins. Extraction: The derived peptide solution

from the Rhopalurus Princeps is extracted using the well-known technique of electric inductance.

The drops are calibrated using a five decimal analytical balance: the results produce an average

of 6.6 ± 1.1 µg per drop. A few drops of distilled water are used to make the derived peptide

flow through the collection test tube.

A 100 ml glass container filled with 100 ml of medical distilled water is used for extraction.

After using electrical stimulation, the drops of scorpion venom are captured in this single 100 ml

sterilized glass container.

Centrifugation: The total amount of derived peptide is centrifuged for 15 minutes at a velocity of

6,000 rpm. The lower solid portion is then separated by decantation.

Filtration: Scorpion venom is a complex mixture of salts, small molecules, peptides, and

proteins. The laboratory filters the venom using Glass Fiber Membrane Filters, 0.80µm, 25mm,

1pk/50pcs to ensure the sterilization and purification.

Therapeutic Dose Preparation: The spectrophotometric analysis is done within a UV range using

a Beckman uv-vis spectrophotometer. The absorbance reading is approximately 278 nm. An

average of 15% of the peptide solution remains in the solid layer.

Different dilutions of the derived peptide solution have similar relative absorbance at 278 nm.

The quantity used for the final product is 0.0035 µg per 120 ml.

The dilution of the concentration for Escozine for Pets™has to be calculated from the portion of

the amount of drops used in the initial extraction from scorpions assuming a 15% loss due to

centrifugation. The absorbance of the initial solution is taken as a parameter to extrapolate the

final dilution.

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Lowry's method for determination of protein concentration

To determine the most therapeutically effective dose concentration of Escozine for

Pets™we use Lowry’s Method.

The Lowry Method (1951) is a colorimetric method of quantitative evaluation of the

proteins. The reagent forms a colored complex with the proteins when added to the

sample; the intensity of color of the resultant solution in proportion to the protein

concentration is measured according to the Lambert-Beer law.

1. MATERIAL AND REAGENTS

a. MATERIALS:

1.

Test-tubes.

2.

Pipettes.

3.

Colorimeter (Beckman)

b. REAGENTS

1.

Reagent A: Na2CO3 to 2 %, NaOH 0.1 M

2.

Reagent B1: CuSO4 × 5H2O to 1%

3.

Reagent B2: sodic-potassic tartrate to 2%

4.

Reagent C: It is prepared at the moment of initiating the essay,

mixing A, B1 and B2 in proportions 50:0, 5:0.5 (in volume)

5.

Reagent Folin-Ciocalteau: commercial reagent diluted to 1/4

6.

Pattern Solution of albumin of bovine whey (2 mg/ml)

2. EXPERIMENTAL PROCEDURE

A pattern curve is drafted, using different volumes of a solution of albumin of

bovine whey (2 mg/ml). The concentrations that the samples have are

determined by interpolation of the absorption values in the curve pattern. The

pipette 0, which only contains distilled water and the reagents, serves as the

target for the adjustment of the colorimeter to zero absorption.

Pattern

(2mg/ml)

Error definition

Water

Target

React. C

Folin dil.

Abs.580nm

0

1.0 ml

--

--

5 ml

0.5 ml

1

0.9 ml

0.1 ml

--

5 ml

0.5 ml

2

0.8 ml

0.2 ml

--

5 ml

0.5 ml

3

0.7 ml

0.3 ml

--

5 ml

0.5 ml

4

0.6 ml

0.4 ml

--

5 ml

0.5 ml

5

0.7 ml

--

0.3 ml

5 ml

0.5 ml

6

0.5 ml

--

0.5 ml

5 ml

0.5 ml

Steps to be followed:

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1. Number from 0 to 6, 10 ml plastic pipettes.

2. Pipette the water quantities, pattern solution of albumin and target solution indicated

in the chart.

3. Prepare the reagent C, from A, B1 and B2.

4. Pipette reagent C to all the pipettes. Mix the content of every pipette and allow it to

rest 15 minutes in darkness.

5. Next, add the reagent of Folin (diluted ¼) to all pipettes, mixing well. Allow to rest 30

minutes in darkness so that the colored reaction develops completely.

6. Read absorptions in the colorimeter to 580 nm. Previously the device is calibrated to

A=0 with the target (pipette nº 0); only the color produced by the proteins is

measured (the color is reduced due to the reagents).

TREATMENT AND DISCUSSION OF THE RESULTS

Obtain the calibration curve representing the absorption levels of the pipettes 0 to 4

opposite to the concentration (or quantity) of protein in every pipette, which is calculated

previously from the information in the chart.

Determine the protein concentration of the target sample, expressing the result in mg/ml

(allow the realized dilutions in the calculation).

Estimation of the protein concentration (Direct reading of the venom in the

spectrophotocolorimeter)

The protein content is estimated from the measurement of the absorption to 280 nm in a

1cm vat, assuming an absorption of 1 mg/ml of raw venom.

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λ

Reading

Lamp

PROG

RS

ABS

% T

UV

8

5

2

VIS

9

6

3

STEP

RCL

BSTP

STOP

REA

CALL

λ

XY

CH

+

-

X

7

4

1

CALL

GOTO

SCA

N

%

0

.

Entr

FUNC

ALFA

OPERATING GRADIENT

Time

Inicial

2.00 min

12.00

min

14.00

min

44.00

min

46.00

min

TABLE #: 6

Curve

Flow

% A

0

0

0

% B

100

100

100

% C

% D

*

6

6

100

0

6

100

0

6

100

0

6

HOME

2

5

8

0

ENTER

1

4

7

3

6

9

.

CLEAR

Refrigeration:

Once the therapeutic dose is determined and the extraction concentrate is ready for

manufacturing, the container is sealed and placed in a refrigerated environment. The

container is kept in that refrigerated environment for thirty minutes until the venom

reaches a maximum of 12C (52F). At this time, the container will be packed into a

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refrigerated compartment that will keep an even temperature range between 12 and

14C (52-58F).

III.

Polarization of Extract and Quality Control of Polarization

Dose preparation before polarization:

All measurement flasks and glass tanks are sanitized in advance.

Upon receiving the purified concentrated extract, the physicist-chemist from the

polarization department measures the required amount of medical grade distilled water

for additional dilution. This dilution is not a final dilution but only for the polarization

process.

Polarimeter test for non-polarized extract: The laboratory

technician separates a small amount of extract and runs

Polarimeter test (Automatic Polarimeter Rudolph (RRA))

This test identifies the atomic movement of the extract (levorotatory or dextrorotatory

movement, and the angular rotation of the atoms as well as the molecular frequency of

the extract. The data is used to compare the molecular activity of the extract before and

after polarization to control and measure the required polarization level for therapeutic

effect.

Polarization: The laboratory technician under the supervision of physicist-chemist then

pours the diluted extract into polarizer’s glass tank. The physicist-chemist then uses the

formula to calculate the speed of the polarizer’s pump and the required quantity of

cycles that is necessary to run the extract through electromagnetic field in order to

reach the required amount of polarization. He then adjusts the power (strength) of

electromagnets, frequency and time on the frequency generator; when all preparation is

completed, the technician starts the polarization process.

Upon completion of polarization, the physicist–chemist checks the polarization level with

an additional polarimeter test.

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ESCOZINE POLARIZATION: PRE-PRODUCTION / POSTPRODUCTION CONTROL

TEST

Blue Line: Spectrum of

the environment

Violet Line: Spectrum of

Non Polarized Escozine.

Red Line: Spectrum of

Polarized Escozine

Blue Line: Spectrum of Laboratory Environment

Violet Line: The spectrum of the Escozine for Pets™ with the mountain (marked by a

cycle) is in the region 3500-3000 cm¯ ¹ wave number.

Red Line: The spectrum of the polarized ESCOZINE FOR PETS™. The mountain is

not present and the spectrum is in the region 2000-500 cm¯ˉ¹. The wave number is

more defined due to the increase of absorbance and the increase of polarization

levorotatory isomer.

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This control test compares the Escozine for Pets™condition before and after the

polarization. When therapeutic level of polarization is achieved, the extract is sent to the

production facility for the final manufacturing process.

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IV.

Manufacturing, Quality Control, Warehouse And Shipment

Receiving: The manufacturing facility of the pharmaceutical laboratory receives purified

and concentrated venom. This concentrate is received in individual containers ranging

in size from 100ml to 0.5 liters.

Manufacturing: The dosimeter of automated filling machinery is calibrated to inject 120

ml of mixture into the sterilized bottles. The correct concentration is determined by the

biochemist depending on the production capacity and order requirements. The

concentrate of scorpion extract is mixed with an excipient of polarized medical-grade

distilled water in a container; the mixing supervisor then monitors every dosimeter

injection of the mixture into the bottles. This procedure disperses the concentrate with

recipient evenly. After the 120 ml bottles are filled with the mixture, bottle caps are

screwed and sealed with plastic shrink wrap neck bands.

In the next step the bottled and sealed product moves by conveyer belt to the labeling

section where the expiration date and lot number are printed onto the labels. Following

this procedure, the bottles are packed in individual cartons with instructions and a 20-30

ml plastic cup and a 1ml plastic dropper. Next is the packaging process where individual

cartons get inserted into transportation boxes. The bulk version of the product is

transferred into a sealed, refrigerated storage room.

Storage/Refrigeration: The bulk container is stored in a dark, refrigerated environment

at the storage facility which is maintained at a temperature between 12 and 14 C (52F

and 58F) while awaiting shipment.

Quality Control: Individual containers of the final product from each and every batch

are collected for quality control. Microbiological tests and other additional tests are

performed to insure the right concentration of the main ingredient is in each batch. The

therapeutic concentration level is tested by using mass spectrometry analyses; the

results are compared to standardized mass spectrometry readings for the product. No

product is shipped until the batch has undergone testing.

Batch Rejection:

A batch of final product is rejected upon the following conditions:

1. Microbiological Test: If one bottle of final product tests positive for bacterial

contamination, then entire batch is rejected.

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2. Incorrect concentration: If more than 3 bottles of the final product show ± 0.05%

difference from the standardized therapeutic concentration by mass spectrometry

analyses, and then the entire batch is rejected.

Shipping: PetLife employees supervise the shipment and handling of all orders to

ensure that the packaging and boxing meets the highest standards.

○○○ DELIVERY TO CUSTOMERS ○○○

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