Downstream Processing of Ferrous Ascorbate

SUBMITTED TO:
Amity Institute of Biotechnology, AUUP

SUBMITTED BY:
Sahil Khan
Enrollment No-A0504116043

SUPERVISED BY-
Dr. Shazia Rashid
Assistant Professor

Amity Instituite of Biotechnology, AUUP

 AMITY INSTITUTE OF BIOTECHNOLOGY
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Enrollment No.: A0504116043
Semester: 7 Section:A
Programme Name: B. tech Biotechnology Batch:2016-2020
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Duration of the project work): 45 days

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Date: 1/9/19 to 15/10/19

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Institute/Industry/University: Amity Institute of Biotechnology

Area of Research: Downstream Processing of Ferrous Ascorbate
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Name of Internal Faculty Coordinator (IFC): Dr Shazia Rashid

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AMITY INSTITUTE OF BIOTECHNOLOGY

PROJECT TITLE : Downstream Processing of Ferrous Ascorbate

COURSE : B. tech Biotechnology

SEMESTER : 7

NAME OF STUDENT : Mohd. Sahil Khan

ENROLLMENT NO : A0504116043

BATCH : 2016-2020

TRAINING PERIOD : 45 days

NAME OF INTERNAL FACULTY COORDINATOR: Dr. Shazia Rashid

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Asst. Director & NTCC Convener

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J- 3 Block, Amity University Campus, Sector – 125, NOIDA – Greater NOIDA Expressway,
NOIDA – 201 313, Gautam Buddha Nagar(Uttar Pradesh, India) Tel. No. - +91 120
4392195, Fax: 0120- 4392947

AMITY INSTITUTE OF BIOTECHNOLOGY

CERTIFICATE

This is to certify that the Seminar Report entitled “Downstream Processing of Ferrous
Ascorbate” submitted to Amity Institute of Biotechnology , Amity University Uttar Pradesh
carried out in partial fulfillment for the award of Btech Biotechnology Degree is a bonafide
work carried out by Ms. Mohd. Sahil Khan Enrolment No A0504116043 of semester 7 from
01/09/19 to 15/10/19.

No part of this project work has been produced elsewhere for any degree or diploma.

Internal Faculty Coordinator

( Name and Signature)

_____________________________________________________________________________________________________
____ J- 3 Block, Amity University Campus, Sector – 125, NOIDA – Greater NOIDA
Expressway, NOIDA – 201 313, Gautam Buddha Nagar(Uttar Pradesh, India) Tel. No. -
+91 120 4392195, Fax: 0120- 4392947
 FACULTY GUIDE APPROVAL

This is to certify the Seminar project entitled “Downstream Processing of Ferrous

Ascorbate” at Amity Institute of Biotechnology is a bonafide work of Mohd. Sahil Khan was
carried out in partial fulfillment for award of B. tech Biotechnology Degree under my
guidance.

This project is original and not submitted for award of any degree at the institution,

Internal Faculty Coordinator

( Name and Signature)

ABSTRACT
Ascorbic acid or vitamin Cis a weak sugar corrosive, white, &soluble. It is same as Glucose.
In natural framework it can be discovered just at low pH however, in impartial arrangements
above it can be found at pH5. It is overwhelmingly found in ionized shape called ascorbate.
Ascorbate is effortlessly &reversibly oxidized to dehrydro-L-ascorbic corrosive, framing the
ascorbyl radical. It is a middle item. Ascorbic corrosive upgrades press retention in the body
by shaping solvent ferrous ascorbate buildings. These are promptly invested in the cell.
Ascorbic corrosive is essential in the improvement of connective tissue cells in our body. It
tweaks neurotransmitter framework in the cerebrum &also fortifies collagen union, which is
the fundamental necessity for muscle& bone arrangement in the body.
We know, Organic acids are building square chemicals& they have a promising worldwide
market. Downstream preparing is critical as far as the creation of natural acids of high virtue.
Aging soups are a mind boggling blend which comprises of cells, spores, wanted product&
undesirable metabolites. These particles must be isolated from the item through a progression
of unit task known as downstream preparing. There are sure maturation factors that influence
downstream handling, for example, Type and nature of smaller scale life forms, partition of
media contaminations and process financial matters. The last imperative advance is that the
maturation soup must be handled and cleansed. These means incorporate Separation of
biomass, cell disturbance, the convergence of stock, item particular filtration, crystallization
and drying.
So, this work is carried out to learn about steps that are carried out in downstream processing
& also to know about how ferrous ascorbate in prepared & utilized in drugs. Properties of
ferrous ascorbate are studied in detail added a brief study is done on API.

INTRODUCTION
Press lack is the most well-known in our populace. It is the single reason for frailty around
the world. The primary reason for press inadequacy sickliness (IDA) in creating nations is
blood lost amid period in premenopausal ladies. India keeps on being one of the nations with
high pervasiveness. Note that an exogenous wellspring of folic corrosive is essential for the
support of typical erythropoiesis in our body.
Ferrous ascorbate folic dangerous reliably called as folate is in reality fundamental in the
whole pregnancy particularly amidst the beginning circumstances of starting. A pregnant
lady should take folate supplements since that can enable her to deny having iron
insufficiency illness which is incredibly fundamental among pregnant ladies. For a pregnant
lady the correct estimation of ferrous ascorbate folic dangerous which ought to be taken in is
5 mg for consistently. There are a gigantic measure of folic dangerous improved sustenance
like the green verdant vegetables, vegetables, citrus characteristic things and juice, entire
wheat pasta and bread, broccoli, peanuts &many more.
Ferrous Ascorbate is a synthetic molecule of ascorbic acid &should be taken as supplements
because it enhances absorption of iron in the body. Ascorbic acid reduces ferric iron to
ferrous iron& it is soluble at neutral pH. It is used in nutraceutical& health care preparations.
It can be made in the form of liquid, capsule & tablet etc.
REVIEW OF LITERATURE
DOWNSTREAM PROCESSING
The downstream handling of ferrous ascorbate is a pharmaceutical procedure of assembling
process driven by both the profoundly utilized process specifically group process and
consistent process, in any case, the persistent procedure may cut some cost and quality
thought. Nourishment and Drug Administration endorsed both the way toward assembling
the Actively Products and Intermediates. Pharmaceutical Companies put a ton of cash in the
Research and Development for better creative medications, versatility and cost decrease.
Downstream processing is usually divided into the following stages:-
1) Solid-Liquid Separation: It is seclusion of entire cells and different fixings that are
insoluble from the way of life media. A few strategies utilized for strong fluid detachment are
buoyancy, filtration, flocculation and centrifugation.
2) Release of Intracellular Products: Sometimes, the required item (vitamins, chemicals)
are situated inside the cells. Along these lines, they ought to be discharged (maximally and in
a functioning structure) for their disconnection and handling. Yeasts, microorganisms,
protozoa and different cells can be upset by concoction, physical or enzymatic strategies.
3) Concentration: Filtrate free from suspended particles more often than not has 80-98% of
water. The required constituent is in little amount minor &water ought to be separated to
accomplish the item focus. Most regularly utilized techniques for concentrating such items
are vanishing, film filtration, fluid extraction, adsorption and precipitation.
4) Purification by Chromatography: The results of maturation are adequately refined by
chromatography. Distinctive sorts of chromatography utilized as a part of downstream
preparing are Ion-trade chromatography, fondness chromatography, Gel-filtration
chromatography, hydrophobic communication &immobilized metal-particle partiality and so
forth.
5) Formulation: It implies the support of security and action of wanted items amid capacity
and circulation. The detailing should be possible by concentrating them with evacuation
water. For certain little particles, it should be possible by crystallization by including salts.
Proteins as a rule require certain balancing out added substances to expand their timeframe of
realistic usability.
 Fig.1. Steps of downstream processing

Purification

Chromatography - Chromatography is the technique of purify protein to purity products,

especially with proteins.Chromatography is important tool on all phase of the Downstream
Processing from the first getting stage to the final polishing stage.
There are many methods of chromatography: -
• Ion trade chromatography (IEX) – It is the strategy for cleaning of proteins and
other charged iotas. Decidedly charged iotas are pulled in to conflictingly charged
solid particles.
• Hydrophobic cooperation chromatography (HIC) – It more often than not isolates
protein particles utilizing the properties of hydrophobicity. In this specific technique,
proteins containing both hydrophobic and hydrophilic locales are connected to a
chromatography section with hydrophobic communications under high salt cradle
conditions.
• Affinity chromatography (AC) – This method is utilized to filter and focus a
substance from a blend to a buffering arrangement, or it can likewise be utilized to
decrease the quantity of undesirable components.
• Gel filtration chromatography (GFC) – It can have used isolates proteins and
peptides in view of size. Particles can go through more prominent or littler permeable
layers by dissemination.
Crystallization
Upkeep of action and dependability of an organic item amid capacity and dispersion is
imperative. The plan of low sub-atomic weight items can be made by concentrating them by
evacuating water. Proteins might be detailed as arrangements, suspensions or as dry powder.
Final fill & storage including sterile filtration
This stage comes after downstream purification and crystallization. This is when the final
product is ready with greatest value. The product which is being packaged mistakenly can
result in safety issues, production failure or loss of expensive product. Therefore, it is
important to maintain the quality&sterility of the pharmaceutical products.

ACTIVE PHARMACEUTICAL INGREDIENT (API)

The greater parts of the medications are comprised of two principle segments which are; the
API, the primary focal fixing, &the excipients which is the substance other than the
medication that aides in conveying the medicine.An active pharmaceutical ingredient (API)
is the component of pharmaceutical drug that is active biologically &excipients are the
components which are inactive chemically substances (eg. lactose or mineral
oil).Pharmaconis the traditional word use for the API which originally denoted to give a
magical substance to drug.

Strength of API’s
Makers utilize certain measures which causes them to decide how solid the API is.
Nonetheless, the standard may fluctuate from brand to mark. For each situation, makers are
required by the FDA who shows the quality of the things, in actuality, patients and in the
expansion investigate office conditions.

Where are API’s made?

The vast majority of the pharmaceutical organizations are situated in the United States,
&numerous API producers are in abroad. The most extreme is situated in Asia, especially in
China &India. A large portion of the organizations are swinging to an outsourcing of API
makers like Dr Reddy's to cut the expenses on representatives, costly gear, &research offices.
AstraZeneca Pharmaceuticals which is one of the most popular pharmaceutical companies is
operating several manufacturing centers in the United States.

Regulations
The nature of APIs is of most extreme significance since it assumes a critical part on the
adequacy and security of medicines. Ineffectively fabricated or traded off APIs have been
found to cause major issues which incorporate sicknesses and may even reason passing.
Notwithstanding when the case is of outsourcing, APIs are liable to controls and oversight
from the nation they are sent to. (Illustration, API producing labs still experience an
investigation by the U.S. Sustenance and Drug Administration).
As prove by the creation of APIs, comprehend that the pharmaceutical enterprises are quickly
evolving. Organizations can never again handle each and every progression of the medication
influencing procedure to like delivering the API to building the container; so with a specific
end goal to eliminate costs and increment benefits; organizations began outsourcing the API
items from remote makers.

CHEMISTRY OF FERROUS ASCORBATE

Ferrous Ascorbate is formed as a result of reaction of ascorbic acid with iron. It is used in
nutraceutical& healthcare. It can be prepared in the form of liquid, capsule or tablet etc.

Molecule Structure
Chemical Properties
 Boiling Point - 415.8°C
 CAS Number - 24808-4
 EINECS Number - 49-7
 Molar Mass - 406.08 g/mol
 Molecular Formula - C12H14FeO12

Physical Properties
 Form - crystalline
 Color - dark brown
 Solubility - Solubility - Ferrous ascorbate is dissolvable in water and is likewise
solvent in DMSO. pH of 1% ferrous ascorbate in water is observed to be 6.73. It is
insoluble in natural in a few solvents like methanol, ethanol, isopropyl liquor
&chloroform.
 Solid state stability - Ferrous ascorbate was observed to be warm steady when it was
kept at 60°C for seven days.
When formulated, it is available in fine dark violet powder; it is a synthetic molecule
of ascorbic acid & iron. This powder is used in the form of tablets, capsules, liquids,
& pellets.

Pharmacology and Biochemistry

 B - Blood & blood forming organs
 B03 - Antianemic preparations
 B03A - Iron preparations
 B03AA - Iron bivalent, oral preparations
 B03AA10 - Ferrous ascorbate

MANUFACTURING OF FERROUS ASCORBATE

The readiness of ferrous ascorbate is when ascorbic corrosive is broken up in water and then
N/10 arrangement of sodium hydroxide is added to it. The arrangement framed is sodium
ascorbate, which is responded with ferric chloride answer for get clear violet arrangement. It
is subjected to vanishing under diminished weight in a CO2 to get ferrous ascorbate. The
ferrous ascorbate contains sodium chloride. The sodium chloride can be expelled by part
crystallization.
The enhanced and modernly attainable process for the readiness of ferrous ascorbate are as
take after wherein ferrous salts are detached and responded with ascorbic corrosive took after
by splash drying procedure to get the ferrous ascorbate with enhanced yield.
Especially, the innovation gives a procedure to the arrangement of ferrous ascorbate
involving the means of:
(a)Reacting soluble base or basic earth metal salt or its hydroxide in a fluid medium with
ferrous or ferric salt, trailed by treatment with ascorbic corrosive to get ferrous ascorbate in
arrangement, wherein the expansion of ferrous or ferric salt and ascorbic corrosive is in any
request;
(b)Filtering the arrangement of step (an); and
(c)Finally, subjecting the arrangement of step (b) to splash drying to get strong ferrous
ascorbate.
The ferrous ascorbate got by the above procedure is of wanted and satisfactory quality,
molecule measure, mass thickness and dampness content.

USES OF FERROUS ASCORBATE / FOLIC ACID

Ferrous Ascorbate is a solution that is utilized for the treatment of anemias which are of
healthful source, pregnancy, earliest stages, Spina bifida in an unborn embryo &Treatment of
megaloblastic anemias because of an inadequacy of folic corrosive and so forth.
Ferrous Ascorbate / Folic Acid improve the patient's condition by performing the following
functions:
 Improving the iron deficiency & absorption of iron from the stomach.

 Acting on megaloblastic bone marrow to produce a normo-blastic marrow.

DESIGN CRITERIA OF PHARMACEUTICAL PRODUCTS

The last game plan of a pharmaceutical thing is incredibly broadened and it includes stages.
The plain first stage is the arrangement exposure organize. In this stage, the API is settled.
Once the API is known, the indicating and assembling of the pharmaceutical thing can be
settled and the procedure can be done effectively.
Other manufactured blends, for instance, excipients are added to the enumerating and
obsession which consequently offsets the API. It also extends its ampleness. Dynamic stages
are incorporated which consolidate layout of the amassing strategy i.e., from pilot plant to
scale up. The last movement procedure for the pharmaceutical thing is settled which can be
taken in with the help of oral systems, for instance, tableting or embodiment. These are the
most broadly perceived and supported choices. Finally, the thing so molded is supported and
promoted. It is set up to be passed on in the market.
Thing layout models and rules can be associated with any of the stages in the pharmaceutical
change process. Typically watched that essentially, various new pharmaceutical things are
found and made by chance using far reaching primer and screw up systems and
strategies.The last development of the solution is in vivo procedure which can be besides
overhauled by outlining out particles that have properties. This enables the medication to be
passed on at the coveted conditions and conditions.

Programming interface Design and Development

The API is reliably outlined to such a degree, to the point that it has the objective of talking
with a specific biomolecule in the patient's cell. These objective biomolecules are engineered
substances and receptors on the cell's surface. The API should in like way have
inconsequential relationship with the regular particles that are not being revolved around.
The soundness and bioavailability of the medication particle is likewise of a pressure while
picking a beneficial API. The repressions made on the execution of a pharmaceutical
influence a restricted course of action to space for the last picked particle.

Structure-Based Drug Design

Exactly when key constraining parts, or ligands, have been perceived through computational
screening, by then comes movement which can be utilized to make the last arrangement
particle. The ligand alone may not effectively arrange the fated TPP of the pharmaceutical
thingThe ligand perceived through receptor-ligand design fills in as a stage to which valuable
social occasions are included which also upgrades the properties of the solution molecule; in
this manner will transform into the dynamic pharmaceutical settling.

Pharmaceutical Formulation Design

Once a medicine molecule has been picked viably, an enumerating for the last
pharmaceutical thing can be pushed ahead. Programming interface setup routinely revolves
around the two essential things and that are quality and selectivity of the picked sedate
molecule which are addressed by a ligand-receptor legitimate. There are various other criteria
that are imperative for the achievement of a pharmaceutical thing. The last thing ought to
have an acceptable prosperity profile which must be appeared on the TPP.

DISCUSSION
Ferrous Ascorbate salts have preferable resistances over the relating feebly acidic ascorbic
corrosive on the grounds that Ascorbates are profoundly receptive cancer prevention agents
utilized as nourishment additives. In pregnant ladies, the occurrence of interminable
obstruction and pyrolysis is marginally expanded. Ferrous ascorbate is molded as a reaction
consequence of ferrous hydroxide and ascorbic destructive. It is a blue-violet powder
containing sixteen for every penny press. It can be typically found in citrus natural items and
various vegetables. Ascorbic destructive is an essential supplement in human eating regimens
and is critical to keep up connective tissue and bone in the body. Ferrous Ascorbate is an iron
supplement, used stay away from squeeze deficiency sickliness. The most broadly perceived
indications saw are impediment and squeamishness. Its normally powerful shape is called
vitamin C, functions as a diminishing master and every so often as a coenzyme in a couple of
metabolic pathways.
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