It was an assignment of mine when i was undergraduate, studying at Gono Bishwabidyalay. this assignment contains: Introduction, Definitions, Unique characteristics, categories, routes, advantages and dis-advantages. On insulin part i focused on: Introduction, different formulations of insulin, injectable insulin preparation, methods of insulin preparation, quality control of insulin, quality control parameter, common quality control tests, packaging and packaging materials..

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Gono Bishwabidyalay
Nolam, Saver, Dhaka
Assignment on Parenteral Products
Submitted To:
Md. Khalequezzaman
Senior Lecturer
Department of pharmacy, GB
Submitted By:
Md. Nazmul Islam Tanmoy
Class roll: 74
Exam roll: 2064
Batch: 32
Semester: 6th
Department of pharmacy, GB.
Course title: Industrial Pharmacy &
Pharmaceutical Technology-II (pharn-3601)

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INTRODUCTION:
• Parenteral preparations are sterile, pyrogen-free liquids (solutions,
emulsions, or suspensions) or solid dosage forms containing one or more
active ingredients, packaged in either single-dose or multi-dose containers.
• The term Parenteral derived from two Greek words 'Para' outside &
'Enterone' intestine.
• Thus are intended for administration by injection, infusion, or
implementation into Parenteral drugs are administrated directly into veins,
muscles or under the skin or more specialized tissues such as the spinal cord.
the body.
Definitions
➢ BP: “Parenteral preparations are sterile preparations intended for
administration by injection, infusion or implantation into human or animal
bodies"
➢ WHO: Parenteral preparations are sterile, pyrogen-free liquids (solutions,
emulsions, or suspensions) or solid dosage forms containing one or more
active ingredients, packaged in either single-dose or multidose containers.
They are intended for administration by injection, infusion, or implantation
into the body.
➢ In short Parenteral preparations are defined as solutions, suspensions,
emulsions for injection or infusion, powders for injection or infusion, gels
for injection and implants.
➢ They are sterile preparations intended to be administered directly into the
systemic circulation in humans or animals.
Unique Characteristics of Parenteral products
• Sterile Particulate-free
• Pyrogen free
• Stable for intended use
• pH - not vary significantly
• Osmotic pressure similar to blood

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Categories of parenteral preparations:
▪ Injections or infusions
▪ Concentrates for injections or infusions
▪ Powders for injection or infusions
▪ Gels for injections
▪ Implants
Routes of Parenteral Administration
• Intradermal (I.D)
• Subcutaneous (S.C)
• Intramuscular (I.M)
• Intravenous (I.V)
• Intra-arterial
• Intracardiac
• Intra-articular (joint)
• Intrasynovial (joint fluid area)
• Intraspinal, Intrathecal (spinal fluid)
Characteristics of parenteral Preparations
• Intravenous (IV) preparations are either:
➢ solutions (in which ingredients are dissolved)
➢ suspensions (in which ingredients are suspended)
• Most parenteral preparations are made of ingredients in a sterile water
medium.
• Some parenteral preparations may be oleaginous (oily).
• Parenteral IV preparations must have chemical properties that will not:
➢ damage vessels or blood cells
➢ alter the chemical properties of the blood serum
• With blood, IVs must be:
➢ iso-osmotic
➢ Isotonic
• Human blood plasma has a pH of 7.4
➢ slightly alkaline
➢ parenteral IV solutions should have a pH that is neutral (near 7)
• Characteristics of parenteral preparations that are important to adjust:

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➢ Tonicity,
➢ Osmolality,
➢ pH are characteristics of parenteral preparations.
➢ It is important that they be adjusted to be as close as possible to the
values for human blood, to prevent damage to blood cells and organs.
Advantages of Parenteral Administration
➢ Fastest method of drug delivery
➢ Viable alternative
➢ Use for Uncooperative patients
➢ Nauseous patients
➢ Unconscious patients
➢ Less patient control
➢ For the patient who can have nothing by mouth
➢ Prolonged action
➢ Correcting serious fluids and electrolyte imbalance
➢ TPN
Disadvantages of Parenteral Administration
• Trained personnel
• Pain
• Difficult to reverse an administered drug's effects
• Manufacturing and Packaging requirements
• Cost
• Needle sticks Injury
Insulin Injection
Introduction:
• Insulin is a peptide hormone, produced by beta cells of the pancreas and is
central to regulating carbohydrate and fat metabolism in the body.
• Insulin is the mainstay of treatment for patients with type-1 diabetes and also
important in type-2 diabetes when blood glucose levels cannot be controlled
by diet and medicines.

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• In diabetes treatment, Insulin is mostly administered by injections, Insulin
pen and Insulin pump
• Mostly referred to as "HUMULIN".
• Its potency, calculated on the dried basis, is not less than 26.5 USP Insulin
Units in each mg;
• Insulin labeled as purified contains not less than 27.0 USP Insulin Units in
each mg, calculated on the dried basis.
• The proinsulin content, determined by a validated method, is not more than
10 ppm.
• NOTE-One USP Insulin Unit is equivalent to 0.0342 mg of pure Insulin
derived from beef or 0.0345 mg of pure Insulin derived from pork.
❖ Different formulations of Insulin:
❖Injectable Insulin Preparation
• Injectable insulin preparations are sterile preparation of insulin (human
insulin, bovine or insulin porcine).
• They contain NLT 90% and NMT the equivalent of 110% of the amount of
insulin stated on the label.
• They are either solutions or suspensions or they are prepared by combining
solutions and suspensions.
• Recombinant human insulin: a form of insulin (trade name Humulin) made
from recombinant DNA which is human insulin.

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Methods of insulin production:
There two main methods exist for the production of recombinant human insulin
from genetically modified bacterial cultures:
Two chain method (both A & B chain are synthesized by separate E. coli plasmid).
Proinsulin method (intracellular or secreted).
The proinsulin method is currently the most efficient method because, single
isolation & isolation steps involved.
A. Innoculum preparation:
➢ According to design process:
- six 200 ml test tubes need to grow for initial culture of proinsulin producing
bacteria
- 1L of tryptic soy broth
- 5g of kanamycin monosulfate
- 5g of the genetically altered E .coli
- Grow for 24 hrs at 37 ° c
- Then placed with a in bioreactor to promote further growth & proinsulin
production.
B. Media preparation:
• The E. coli must be placed in a mixture containing the essential building
blocks for growth, including carbon, nitrogen, phosphorus.
1. Bioreactor with 23L total volume & 16L working volume.
2. The 1L of E.coli & depleted growth medium is mixed with 9L of growth
media in bioreactor. (For C- glycerol & yeast extract, N- A. sulphate &
thiamine,)
3. Buffer: pota. dihydrogen phosphate & dipota. phosphate.
4. pH: at 7.
5. 0ther nutrients included in the broth are Na. citrate, Mg. sulphate, a
trace element solution, & a vitamin solution.
6. The oxygen tension is also monitored to be kept at a tension level of
30%.

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C. Fermentation process of insulin production:
1. Fermentation: The first of the process is to grow enough of the
proinsulin producing E. coli bacteria so as to acquire a sufficient amount
of insulin per process.
- In order to do this an original amount of E. coli cells containing the plasmid
for proinsulin production will be grown in test tubes containing tryptic soy
broth & kanamycin mono-sulphate.
- All of the accomplished by placing the original growth mixture into a
bioreactor in which the parameters can be controlled for maximum cell
growth & insulin production.
- Within a bioreactor the temperature, PH, foam, & feed can be controlled
automatically to yield maximum result.
- Equipment: BioNet Reactor.
- Reagents Involved:
➢ 1L inoculation solution
➢ 25mL NH3
➢ 30mL H3PO4
➢ 100mL 87% glycerol feed
➢ 25g (NH4) 2S04
➢ 30G KH2PO4 * H2O
➢ 20G KHPO4 * 2H2O
➢ 5g Na3-citrate
➢ 10g yeast extract
➢ 0.7g thiamine
➢ 10ml trace element solution
➢ 6.5mL vitamin solution

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➢ 10ml adecanol -109 (antifoam)
➢ 10ml B-indole acrylic acid
➢ Water up to 10L total
- Parameters:
➢ 10L total working volume
➢ 31 hours growth phase
➢ pH 7
➢ 37 C
2. Centrifugation: Here used for four times throughout the process.
- Equipment: Avanti J-HC
i) Cell isolation: The first steps in insulin production is the isolation of
the bacterium containing proinsulin inclusion bodies, this process also
called cell harvesting. cell harvesting could be used including
filtration & centrifugation.
Parameter: 7500g for 10 min.
ii) Indusion body separation: Proinsulin from rest of cell debris by
centrifugation or reverse osmosis.
Parameter: 15000g for 30 min.
iii) Additional Separation: for removal of reagents.
Parameter: 17700g for 33 min.
iv) Volume reduction: Prior to downstream purification.
Parameter: 17700g for 33min.
3. Cell disruption- Homogenization: By high pressure or alkali treatment.
- Equipment: Nano DeBEE Electric Blade-type Homogenizer.
- Parameter: homogenizer supplies pressure of 45,000 PSI & Flow rate:
150ml / min (3L E.coli cell mixture for 20 min)
4. Centrifugation: Same as previous.

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5. Solubilize inclusion bodies: Solubilization of inclusion bodies is carried
out by addition of a denaturing agent such as urea or guaminidinium-
HCL (GdmHCL) .these agent denature the fusion proteins composing the
inclusion bodies.
Parameter: Stirring for 6hr at 37 C 8M Urea added.
6. Sulfitolysis: Sulfitolysis involves addition of -SO, groups to the reduced
sulfur residues on cysteines of proinsulin polypeptides, preventing the
formation of potentially incorrect disulfide bonds during the
solubilization and early purification steps prior to correct refolding of the
proteins.
. Incorrect disulfide bond formation during the solubilization and
renaturation processes of proinsulin production accounts for a significant
decrease in percentage yield.
Parameter:
- Performed during 6hr solubilizing step
- 0.8M Na2SO4
- 0.3M Na2SO4 * 2H2O
7. Centrifugation: Same as previous.
8. Dialysis: Effectively removes denaturants (urea, beta-mercaptoethanol or
DDT) & dissolved contaminants (buffer-tris-HCL, & solubilizing
agents).
Equipment: Spectra / par 1 membrane, MWCO 6-8000.
Parameters: 4 repetitions
Washing buffer: 10mM Tris-HCI (pH 8).
9. Renaturation: To maximize correct bond formation.
Parameters: Stirring for 20hr at 4 C
1M glycine NaOH (pH 10.5+)
18: 1 molal ratio of B-mercaptoethanol to fusion protein
10.Centrifugation: same as previous.
11.Purification:
i). Affinity chromatography: isolation of proinsulin peptide from fusion
protein, further purification is required to produce an insulin product pure
enough for patient use.
Equipment: IgG-Sepharose column

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SDS-PAGE
Superdex 75 PC3.2 / 10
Parameters: 0.3M acetic acid added until pH of 8 is reached
Ammonium acetate
200mM sodium phosphate
Quality control of Insulin
• After every one of the many steps of purification, scientists check and
double-check the insulin's purity.
• Even the packaging process is super-tightly regulated, as each vial of insulin
is photographed from many angles.
• There are many quality control tests performed at manufacturing site
according to its type of formulation.
Quality Control parameter for Injectable Insulin preparation
1. pH It should be 6.9 to 7.8 otherwise as per specific monograph.
2. Insulin in the supernatant
- should be NMT 2.5% of the total insulin content, and insulin content of
supernatant liquid (s) is determined by chromatographic method.
- % Insulin = 100S / T (T: - total insulin content determined by assay)
3. Impurities with molecular masses greater than that of insulin
- It is examined by size-exclusion chromatography.
4 Related proteins It is examined by liquid chromatography.
5 Total Zinc It is determined by atomic absorption spectroscopy.

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- Prepare test and reference solutions and measure absorbance at 213.9 nm
using a zinc hollow cathode lamp or air-acetylene flame as source of
radiation.
6. Bacterial endotoxins should be less than 80 IU per 100 IU of insulin.
7. Assay
examine by liquid chromatography. Mostly, it is carried out by High
Performance Liquid Chromatography (HPLC).
- Test solution: It is prepared by adding 4ul of 6 M HCL per ml of preparation
and with an additional dilution by 0.01 M HCL.
- Reference solution: It is prepared by dissolving insulin in 0.01 M HCL to get
conc. 4 mg / ml and then addition of 1ml of bovine insulin and 1ml of
porcine insulin.
8. Sterility
- Sterility or freedom from presence of viable microorganisms is a strict,
uncompromising requirement of an injectable dosage form.
- Sterility test is performed by following tests,
1. Pyrogen Testing
• LAL test (Gel clot test and Chromogenic test)
the result is expressed in EU / ml.
• Rabbit pyrogen test
Insulin zinc forms ppts. in sterility test media. Ascorbic acid at 1% in
0.1% peptone (% w/v) dissolves protamine zinc and insulin zinc in no
more than one minute without harming organisms.
2. Particulate Matter Testing
• Visual Inspection Test
(i) Manual Methods
- off-line Inspection (Personnel)
(ii) Automatic Methods
- Auto scan System
- Electronic Particle Counters
9. Stability Test
The stability studies are done at 5 ° C (refrigerator) and at room temperature (20 -
25 ° C) under the following conditions.
• In acidic medium (0.01M HCI, pH 2.0) and at room temperature.
. In acidic medium (0.10M HCI, pH 1.0) and at room temperature.

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• In basic medium (0.10M NaOH, pH 13.0) and at room temperature
• In phosphate buffer medium (0.10M, pH 7.4) and at room temperature.
10. Package Integrity Testing
Package integrity is the measure of a package's ability to keep the product in
and to keep potential contamination out.
• Package Integrity Tests:
- Acoustic Imaging
- Ultrasonic Imaging
- High Voltage Leak Detection
- Noninvasive Moisture and Oxygen Analysis O Residual Gas Ionization Test
- Residual Seal Force
- Microbial Challenge Test
- Visual Inspection
- Vacuum / Pressure Decay
- Weight Change
- Bubble Test
- Gas Tracer Detection
- Helium Mass Spectrometry
- Liquid Tracer Test
Common quality control tests for Insulin preparations.
1. Inertness of preparation
It is checked by Capillary electrophoresis. Basically it is used to predict
protein interaction with the capillary wall or with other protein.
2. Structural analysis of Protein
It is analyzed by hydrolysis, pre and post column derivatization, Aufomated
Edman sequencing.
3. Validation Parameters
▪ Linearity Three samples from each of the concentrations (10-100 ug /
ml) used for construction of standard curve and for checking
Linearity.
▪ Precision
Six samples from the concentration range (10-100 ug / ml) is used for
construction of standard curve and analyzed in one day or six different
days in order to evaluate intra-day or inter-day variations (Precision)
respectively

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▪ Range
It is taken between 10 - 100 µg / ml.
▪ Accuracy
The accuracy experiment is performed on four different
concentrations (each injected three times) covering the linear range.
▪ LOD (Limit of Detection)
▪ LOQ (Limit of Quantitation)
▪ Sensitivity
Packaging and Packaging materials
Glass containers are used as insulin vial because they offer some
important benefits that other materials don't offer. They are formed by
blowing, drawing, pressing, and casting. Some advantages of glass
containers are there that make them fantastic option for pharmaceutical
uses.
- They are easy to sterilize with heat.
- Colored glass has the ability to protect its content from certain wavelengths
which include the ultraviolet rays of the sun.
- They are chemically inert and will not react with their content.
- They are impermeable to water and air and that makes them a great storage
facility for drugs.
- They are transparent so, their content can be seen without opening them.
It is also important to let you know that not all glass containers are good for
pharmaceutical use. The factors considered when selecting glass containers are
sensitivity to calcium and barium ions, thermal expansion properties, hydrolytic
resistance, and limited alkalinity. This is why glass containers are categorized
into different types for pharmaceutical uses.
Types of Glass Containers
Glass containers are classified into Types I, Il, III, and IV.
Type I Glass Containers
This type of glass contains 10% of boric oxide, 80% of silica, and small
quantities of both aluminum oxide and sodium oxide. The boric oxide in it
makes it highly hydrolytically resistant and chemically inert. In addition, its
coefficient of expansion is very low and high its thermal shock property is quite

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high. Due to its characteristics, a Type I glass container is great for packaging
materials for a lot of parenteral and non-parenteral preparations. It can also be
used to store strong alkalis and acids.
Type II Glass Containers
This is similar to Type II containers. In fact, a Type II glass container is
regarded as a modified Type II container. Type Il containers have high
hydrolytic resistance. Type Il glass containers are actually Type III containers
whose inner surface have been treated with sulfur. This treatment helps to
prevent weathering from the containers. Type II glass generally has a lower
melting point than Type I glass so it is much easier to mold. Type II glass
containers are suitable for storing neutral aqueous preparations and acidic
preparations whether they are parenteral or non-parenteral.
Type III Glass Containers
This type of glass containers is made of 10% calcium oxide, 15% sodium oxide,
and 75% silica. They also contain negligible amounts of aluminum oxide,
potassium oxide, and magnesium oxide. While magnesium oxide reduces the
temperature required to mold the glass, aluminum oxide improves its chemical
durability. This type of glass container is used for packaging non-parenteral
preparations and for packaging certain parenteral products.
Type IV Glass Containers
This type of glass containers contains general- purpose soda lime and they have
low hydrolytic resistance. This category of glass containers is the best for
products that are meant to be autoclaved because the rate of erosion reaction of
the glass containers will be increased. Type IV glass containers are used for the
storage of oral dosage forms and topical products.
In summary, glass containers are classified into Types I, II, III, and IV for
pharmaceutical uses but type II is the best suitable material for insulin.
CLOSURES:
Rubber consists of long chain polymers of isoprene units linked together in the
cis-position. Hevea braziliensis is the most important source of rubber. Its latex
contains 30% to 40% of rubber in colloidal suspension. Usually closures for
parenteral products are made from

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1. Natural rubber
2. Synthetic rubber & Characteristics of good pharmaceutical rubber:
A good pharmaceutically used rubber consists of the following criteria:
• Should show good aging property.
• Should have satisfactory hardness and elasticity.
• Should have resistance to sterilization conditions.
• Should not affect by moisture and air.
• Should have low permeability to moisture and air.
• Should have negligible release of undesirable substances.
• Should have negligible extraction of injection ingredients.
CONCLUSION
- Quality control should be a fundamental segment Of parenteral products
manufacturing.
- All of the 5 basic tests which are performed are essential and have its own
importance in parenteral production.
- All of these tests ensure that product meet its quality which has been judged
to satisfactory also.
- Each test is unique and provides detailed assessment of quality control for
parenteral products.