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
Stages of Production
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Production Procedures
Escozine for Pets has 4 major production steps.
1.
2.
3.
4.
Collection of Scorpions from the Scorpion Reservation.

Extraction of venom, purification and therapeutic dose preparation.
Polarization of extract and quality control of Polarization
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 Corps 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 g 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, 0.80m, 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 g 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 Lowrys 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.
Error definition
Water
Pattern
(2mg/ml)
Target
React. C
Folin dil.
1.0 ml
--
--
5 ml
0.5 ml
0.9 ml
0.1 ml
--
5 ml
0.5 ml
0.8 ml
0.2 ml
--
5 ml
0.5 ml
0.7 ml
0.3 ml
--
5 ml
0.5 ml
0.6 ml
0.4 ml
--
5 ml
0.5 ml
0.7 ml
--
0.3 ml
5 ml
0.5 ml
0.5 ml
--
0.5 ml
5 ml
0.5 ml
Abs.580nm
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 ) 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 n 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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PROG
Lamp
RS
ABS
STEP
RCL
BSTP
STOP
CALL
GOTO
FUNC
Reading
ALFA
%T
REA
CALL
XY
CH
+
X
7
4
1
UV
8
5
2
SCA
N
OPERATING GRADIENT
Time
Flow
%A
Inicial
0
2.00 min
0
0
12.00
min
100
14.00
min
100
44.00
min
100
46.00
min
%B
100
100
100
%C
VIS
9
6
3
Entr
TABLE #: 6
Curve
*
6
6
%D
1
4
7
CLEAR
HOME
2
5
8
0
ENTER
3
6
9
.
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 polarizers glass tank. The physicist-chemist then uses the
formula to calculate the speed of the polarizers 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 physicistchemist 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 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. 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
supervise the shipment and handling of all orders to
PETLIFE
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ensure that the packaging and boxing meets the highest standards.
DELIVERY TO CUSTOMERS
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