Downstreamprocessing of cephalosporins and aspartic acids in industries

2. • A bioprocess is specific process that uses complete living cells or their components to obtain
desired products.
• It can be separated into upstream processes and downstream processes
Upstream processing Downstream processing

3. • Recovery and purification of biosynthetic products
• It is divided into four major processes
– Removal of insolubles
– Product isolation
– Product purification
– Product polishing

4. • Used to treat infectious diseases
• Destroy pathogenic organisms or inhibit their growth at concentrations low
enough to avoid undesirable damage to the host.
• ANTIBIOTICS : Anti – against & bios – life
• These are products of secondary metabolism
• 1904 - “MAGIC BULLET” by Paul Ehrlich
– Found Tryptan red active against Trypanosome
– With Sahachiro Hata found Arsphenamine effective against Syphilis
– Later in 1910, Arsphenamine was sold under name of Salvarsan
• 1920- Alexander Flemming discovered Lysozyme in tears
• 1927- Gerhard Domagk discovered Prontosil red against streptococci and
staphylococci
• 1928- Penicillin by Alexander Flemming
• 1939- Sulfa drugs by Jaques and Therese
• 1944- Streptomycin by Selman Waksman

5. • Currently 8000 antibiotics are known
• Each year around 300 new antibiotically active compounds are detected, of which
30-35% are antibiotics
• Only 123 antibiotics of bacterial origin are produced by fermentation
• Only chloramphenicol, phosphonomycin and pyrrolnitrin are produced
synthetically
• Significance for the strain is unclear
• Antitumor antibiotics
• Antibiotics for plant pathology
• Antibiotics as food preservatives
• Antibiotics used as animal growth promoters and in veterinary medicine
•Antibiotics as tools in biochemistry and molecular biology

7. • Any of various broad-spectrum beta- lactam antibiotics closely related to the Penicillins, that were
originally derived from the fungus, Cephalosporium acremonium.
• They contain a dihydrothiazinering with D aminoadipic acid as acyl moiety.
• It is also produced by Emericellopsis and Paecilomyces.
ACTION: Inhibitors of peptidoglycan synthesis, Activate cell wall lytic enzymes
COMMON USE: In surgical procedures- to reduce the risk of post- operative infections.
FIRST GENERATION - Cefazolin,Cephalexin
Spectrum: Most G (+)ve cocci (Streptococcus, S. aureus), E. coli, proteus, Klebisella
Use: S. aureus infection, surgical prophylaxis
SECOND GENERATION – Cefoxitin, Cefuroxime, Cefaclor, Cefprozil
Spectrum: Mainly effective gram negative bacteria, modest activity against gram positive bacteria
Use: Primarily for upper & lower respiratory tract infections
THIRD GENERATION – Ceftriaxone, Cefotaxime
Spectrum: enhanced G (–)ve activity
Use: Meningitis, highly resistant & multi drug resistant Streptococcus along with vancomycin
FOURTH GENERATION - Cefepime
Spectrum: Active against Streptococcus, staphylococcus, pseudomonas aeruginosa & aerobic G –ve

8. • Its molecule is transformed by removal of an aminoadipic acid side chain to form 7-α
aminocephalosporanic acid (7-ACA), which is further modified by adding side chains
to form clinically useful broad spectrum antimicrobials

10. • 13 therapeutically important semisynthetic
cephalosporins are commercially produced.
• These have been synthesized by chemical splitting
to form 7 aminocephalospioranic acid (7-ACA) with
subsequent chemical acylation as well as by
modification on the C-3 site.
• Complex media with Corn steep liquor, meat meal,
sucrose, glucose and ammonium acetate are used in a
fed batch system at ph 6-7 and temperature 24-28° C
• Recently chemical synthesis of cephalosporin by ring
expansion of penicillin has been developed.
• Eg. Use of pennicillin V to produce oraspor, an orally
active cephalosporin.

11. • Extraction of the product is challenging task
• Done in several stages and many of these steps are expensive and generate large
quantities of waste water and other chemicals.
• Almost all the known processes for commercial scale extraction and purification
are based on low-yield operation because of the unfavorable physical properties of
the β-lactams
• Difficulties arise in the case of cephalosporin’s which are of highly hydrophilic
possessing zwitterionic properties and have very small differences in polarity.

12. 1. SOLID PHASE EXTRACTION
2. LIQUID PHASE EXTRACTION
1. HPLC
3. LIQUID MEMBRANE EXTRACTION
1. Emulsion liquid membrane
2. Supported liquid membrane
4. MEMBRANE SEPARATION
1. Ultrafiltration
2. Microfiltration
3. Reverse osmosis
5. AQUOUS TWO PHASE PARTIONING

13. •The purification and recovery of harvest cephalosporin C broth begins with the rapid chilling of the
active broth to 3–5C followed by removal of the mycelial solids either by filtration or by
centrifugation.
•The active broth contains not only the desired cephalosporin C component, but also small
quantities of the biosynthetic precursors, penicillin N, deacetylcephalosporin C and the degraded
cephalosporin C product, compound X.
1. SELECTIVE PRECIPITATION AND PURIFICATION
2. SEPERATION AND PURIFICATION BY CHROMATOGRAPHY
1. Thin layer and paper chromatography
2. Ion exchange chromatography
3. Hydrophobic column
4. Gel filtration
5. High performance liquid chromatography
3. ELECTROPHORETIC TECHNIQUES
1. Polyacrylamide gel electrophoresis
2. Isoelectric focussing

14. • Involves electrophoresis in very narrow bore tubes, that reduce problems from
heating effects
• Because of small diameter, there is a large surface to volume ratio, which gives
enhanced heat dessipation
• 2 important equations:
– t = L2/μV
– N = μV/2D
– Where
• t – migration time of a solute
• L – length of tube
• μ - Electrophoretic mobility
• V – voltage
• D – solute diffusion coefficient
• Tube length plays no role but it has an important influence on migration time and
hence analysis time.
• High separation efficiencies are achieved at high voltages

16. • Separation by EAC is carried out in a long ribbon like multicompartment
electrolyser separated by membranes, in which two central components are used
for packing the gel matrix and for sample loading respectively.
• Next to the central compartments are the elution compartment and the electrode
compartments.
• The electric field is applied perpendicular to the fluid flow in the compartments.
• Adsorption and desorption steps both be carried out in the presence of electric
field, which transports the target components into the gel compartment for
adsorption and the impurities in the elution compartment for washing.
• After the adsorption step, an elution solution is introduced in the and the product
is released from the matrix and washed out.

21. • It is an α-amino acid with the chemical formula HOOCCH(NH2)CH2COOH.
The carboxylate anion of aspartic acid is known as aspartate.
• The L-isomer of aspartate is one of the 20 proteinogenic amino acids, i.e.,
the building blocks of proteins.
• Its codons are GAU and GAC.
• Aspartate is non-essential in mammals, being produced from oxaloacetate
by transamination.
• In plants and microorganisms, aspartate is the precursor to several amino
acids, including : methionine, threonine, isoleucine, and lysine.

22. • It is used in parenteral nutrition
• As cosmetics incredient
• As ingredients for cell culture media
• Clinicians include aspartic acid in some natural programs for depression
and immune function.
• Aspartic acid aids in energy production from carbohydrates and in RNA and
DNA formation.
• Aspartic acid aids in liver detoxification from drugs and chemicals.
• Aspartic acid increases resistance to fatigue.
• Found in its highest quantities in the brain, aspartic acid increases
neurologic activity.
• Aspartic acid forms aspartame when it’s combined with phenylalanine.
Aspartame is the commonly used artificial sweetener that can be a mild
irritant to the nervous system.

23. • The amino acids are the second most important category, after antibiotics,
with fermentation products exhibiting the highest growth rates.
• A column packed with immobilized Escherichia coli cells entrapped in a
polyacrylamide gel lattice is used for continuous production of L-aspartic
acid from ammonium fumarate.
• Annual production is 4000 metric tons per year

25. • Centrifugation is used to separate cell material
• Amino acids are obtained after acidification through
– Precipitation at the isoelectric point
– Thin layer chromatography and paper
chromatography
– HPLC
– Ion exchange chromatography
– Extraction with organic solvents.

26. • The isoelectric point, sometimes abbreviated IEP, is the pH at
which a particular molecule carries no net electrical charge.
• IEP of aspartic acid is 2.7
• It is least soluble when the pH of the solution is at its IEP.
• So, it can be precipitated out by using buffer of pH – 2.7
• 1M glycine buffer can be used

27. • All purification steps are carried out at 0-4 °C.
• The sample is applied to a cation exchange chromatography column
containing SP Sepharose HP is equilibrated with 10 mM sodium phosphate
buffer (pH 7.0).
• The column was washed with two bed volumes of the buffer, and
adsorbed proteins were eluted at a flow rate of 5 mL/min with a linear
gradient (500 mL) from 0 to 250 mM NaCl in 10 mM sodium phosphate
buffer.