CEPHALOSPORINS

Group 3
Pharmaceutical Biotechnology

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 Group member
Vương Mỹ Hảo BTBTIU14063

Dương Thị Cẩm Tú BTBTIU14264

Nguyễn Bùi Bảo Trân BTBTIU15068

Nguyễn Thị Hạnh Duyên BTBTIU15101

Tô Ngọc Cát Tường BTBTIU15133

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Introduction

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 CEPHALOSPORINS
• Cephalosporins are β – Lactam antibiotics, that are closely related
to both structurally and functionally to penicillins, derived from
cephalosporin – C.

• Cephalosporins were first isolated from culture of Cephalosporium

acremonium, a fungus, by an Italian scientist Giuseppe Brotzu.

• Cultures of cephalosporium acremonium inhibited the growth of a

wide variety of gram – positive and gram – negative bacteria,
especially, effective against Salmonella typhi (typhoid fever) which
had beta – lactamases.

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CEPHALOSPORIUM ACREMONIUM

Culture (Colonies) Microscopic view

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Production

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Trace elements

• Essential vitamins:

• Aerobic conditions i.e., enough dissolved oxygen supply around 40% or above. • Appropriate
buffer for maintaining optimum pH. Small changes in the pH can easily be detected by the pH
meter.

• Optimum temperature maintained by flowing water through the jacket to promote fast growth of
the fungus.

• Silicone oil to control foaming where necessary.

• Moisture.

Chemical inhibitors and blockers to prevent or inhibit the interaction between crude cephalosporin c
and other chemical compounds which may be structurally similar or dissimilar to crude
cephalosporin c and accumulates in the liquid broth at the end of the fermentation reaction

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Production Process
• Cephalosporin c production on the fermentation medium at 28 ° C for 144 hours,
characterized by the rapid consumption of sucrose at the beginning to form biomass.
(pH 7.2, 28°C, dissolved oxygen above 30% saturation)

• Carbohydrate is depleted by the slow consumption of sucrose

• When most of the Cephalosporin c is produced (idiophase): cell growth is observed
insignificant.
• Maximum cephalosporin c has been obtained at 120 h of fermentation while highest cell
growth occurs at 42 h of fermentation.
• Cephalosporin c is separated from the liquid broth by the use of a number of separation and
distillation techniques
Analysis: The dry cell weight was estimated by centrifugation of 10 ml of fermentation broth,
washed twice with distilled water, recentrifuged and kept for drying at 80°C till the constant
weight.
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Strain improvement for cephalosporin production

• Acremonium chrysogenum strain improvement program based on the transformation with

cephalosporin biosynthetic genes was carried out to enhance cephalosporin C production.

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Microbial strains, plasmids and microbiological procedures

• A. chrysogenum strains B and C, two cephalosporin

overproducing strains
• Escherichia coli DH5α was the recipient for high-frequency
plasmid transformation.
• pBluescript I KS (+) and pBC KS (+) were the plasmids used for
subcloning and sequencing.
• pAN52.1 was the source of Pgpd and TtrpC, Pgdh was obtained
from pALP30 , the aphI gene was from pPIC3.5K and nptII gene
from pBI121.
• A. chrysogenum transformants were selected on TSA sucrose
plates supplemented with 3 μg ml−1 phleomycin (Cayla) or 7 μg
ml−1 G418 after incubation at 28 °C for 10 days.

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DNA manipulations, PCR, Southern and Northern analysis

• DNAs of A. chrysogenum were purified as previously described and

used as templates for PCR reactions

• primers to amplify an internal 875 bp fragment of the gene of A.

chrysogenum
 AS28 (5′-CGCGAGGGTGCATCGCAACG-3′)
 AS29 (5′-GTCCAGGACGATACCGGTCG-3′)

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Southern analyses:
DNAs were digested with BamHI, HindIII and SalI, blotted to a nylon filter and hybridized with the
following probes of A. chrysogenum:
• 2.95 kb BamHI internal to pcbAB gene encoding ACVS
• 2.25 kb EcoRI/XmnI including a portion of pcbC gene encoding IPNS
• 0.5 kb SalI internal to cefEF gene encoding DAOCS/DACS
• 2.0 kb HindIII including a portion of cefG gene coding for DAC-AT

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Northern analyses:

A 0.8 kb SacI probe including a portion of the actA gene encoding

γ-actin was used as a control of the RNA quantity

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RESULT AND DISCUSSION
CONCLUSION

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• Cephalosporin (CPC), deacetylcephalosporin (DAC), deacetoxycephalosporin
(DAOC), and penicillin N and isopenicillin N (PenN + IPN)
Cephalosporin C production was improved and the accumulation of the
biosynthetic intermediates DAC, DAOC, IPN and penicillin N decreased in
transformants B1 and C1, which have an extra copy
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of the cefEF and cefG genes
RESULT AND DISCUSSION
• Effect of methionine:
Increased methionine concentration (up
to 4.0 g/l) → cephalosporin C
concentration increases
Optimum concentration of methionine
stimulates fungal differentiation and
increases the antibiotic yield.

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 RESULT AND DISCUSSION
• Effect of methionine:
→ Hence it is reported that 0.4% of methionine concentration is optimum for the
synthesis reaction

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RESULT AND DISCUSSION
• Effect of ammonium sulphate:
Increased dry cell weight → increases cephalosporin C production
But its high concentration → decreases the rate of synthesis by
interfering in mycelial morphological differentiation

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RESULT AND DISCUSSION
• Effect of C/N ratio:
C/N ratio of 5.33 to 8.0 → increases the synthesis rate of cephalosporin C.
Sucrose accumulation in the broth → inhibits the synthesis of enzymes
accountable for Cephalosporin C formation.

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RESULT AND DISCUSSION
• Increased oral absorptivity and increased cephalosporin c
stability by cleaved blocking groups for the carboxylic acid in
the cephalosporin synthetic structure
• Improve the bioreactor layouts to improve their performance
by ensuring optimum reaction condition for fermentation to
produce high quantities of cephalosporin c and maintaining
stable reaction kinetics.
• This well engineered bioreactor also ensures efficient
utilization of all the reaction components during fermentation
process
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RESULT AND DISCUSSION

• Easy detection of the geometric phase of fermentation during which

fungus growth is at its peak and maximum cephalosporin c product is
obtained, can be identified.
• The fermentation reactor can also be integrated with automated or semi-
automated distilling equipments for the separation of the crude
cephalosporin from the liquid broth much easily and efficiently in less time

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 CONCLUSION

• Cephalosporin C is one of the mostly used

antibiotic worldwide with broad activity
spectrum
• Used against numerous Gram negative and
Gram positive bacterial strains for the
treatment of various bacterial associated
diseases.

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DNA VACCINE
Group 3

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 Content
• About HPV vaccine
• The pros and cons of the HPV vaccine
• Ingredients
• Production of HPV vaccine
• Advandges of Baculovirus expression system
• How is the HPV vaccine given?
• How does the HPV vaccine work?

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 About HPV vaccine
• HPV vaccines are vaccines that protect against infection with human
papillomaviruses(HPV).
• Cervarix is a vaccine against certain types of cancer-causing human
papillomavirus (HPV).
• Cervarix is designed to prevent infection from HPV types 16 and 18, that cause
about 70% of cervical cancer cases.
• Cervarix contains AS04, a proprietary adjuvant that has been found to boost
the immune system response for a longer time.
• Consists of 3 doses of 0.5 mL each, by intramuscular injection at 0, 1 and 6
months.
• Cervarix has shown to be 92% effective and effective for more than 4 years.

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The pros and cons of the HPV
vaccine
• Pros
• The HPV vaccine can protect against HPV types 16 and 18, both of which can
lead to certain cancers.
• Some vaccines can also protect against strains known to cause genital warts.
• Cons:
• Mild to moderate side effects can include: slight fever, headache, fatigue, muscle
pain,….
• The HPV vaccine protects against some types of HPV-related cancers, but not all.

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 Ingredients
• The active components of the vaccine are:
• Human Papillomavirus type 16 L1 protein 20 micrograms
• Human Papillomavirus type 18 L1 protein 20 micrograms
• AS04 adjuvant, containing: 3-O-desacyl-4'- monophosphoryl lipid A (MPL) 50
micrograms adsorbed on aluminium hydroxide, hydrated (Al(OH)3) 0.5
milligrams Al 3+ in total

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Production of HPV vaccine
• Cervarix is created using the L1 protein of the viral capsid. L1 protein is in the form of non-
infectious virus-like particles (VLPs) produced by recombinant DNA technology using a
Baculovirus expression system which uses Hi-5 Rix4446 cells derived from the
insect Trichoplusia ni.

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Production of HPV vaccine
Step 1: Amplification of seed recombinant Baculovirus
Step 2: Extraction of recombinant Baculovirus inocula
Step 3: Infection of Trichoplusia ni Hi-5 production cell lines in fermenter
Step 4: L1 proteins are extracted by methods of osmotic shock
Step 5: Sterile filtration of L1 VLP
Step 6: Combination of HPV 16 and 18 L1 proteins
Step 7: Formulation, filling and packaging

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Production of HPV vaccine
Purification:
• The cell lysate may be subjected to a microfiltration and ultra-filtration process
• Cation- exchange chromatogrophy
• Hydroxyapatite Chromatography:
• Hydroxyapatite as the column medium
• Elution with a buffer solution containing phosphate anion - removes a large amount of
contaminants from a partially purified cellular lysate
• Removes contaminating biomolecules, including DNA, lipids and proteins.

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Production of HPV vaccine
Formulation:
• The L1 proteins
• AS04 adjuvant system composed of- aluminium hydroxide- 3-O-desacyl-4.-
monophosphoryl lipid A (MPL)
• The MPL immunostimulant: a detoxified derivative of the lipopolysaccharide
ofthe gram negative bacterium Salmonella minnesotaR595 strain.

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Quality control
Cell seed and banking:
• The MCB, WCB, MS and WS and End of Production Cells (EPC) are
tested for identity, purity and safety
• The Assays are:
• Tumourogenic Potential
• Adventitious Agents
• Genetic Stability
• Specific Viral Contaminants

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Quality control
Process control and validation:
• The 3 unit-steps of the drug substance production process are tested for
consistency:
• The recombinant HPV-16/18 baculovirus inoculum production process
• The L1 single harvest production and L1 extraction
• The L1 antigen purification process.

Manufactoring process development

• The final process purified bulks, are assessed by subjecting each type of
materials to a series of physico-chemical analyses and immunogenic property
tests.
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 Advandges of Baculovirus
expression system
• High levels of heterologous gene expression
• Grows well in suspension cultures, easy for large- scale production
• Safe (restricted to infection of invertebrate species)
• Cheaper as Hi-5 Rix4446 can be cultured in serum free medium

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 References
• https://bpac.org.nz/BPJ/2011/december/docs/bpj_41_cephalosporins_pa
ges_22-28.pdf
• https://pdfs.semanticscholar.org/27f9/7dbdf733dac96aeabbcba493e3a11
b64493e.pdf
• https://medimoon.com/2013/05/classification-of-cephalosporin-antibiotics/
• https://www.verywellhealth.com/what-are-cephalosporins-1124176
• http://www.hpvvaccine.org.au/the-hpv-vaccine/how-does-it-work.aspx
• https://www.cancer.gov/about-cancer/causes-prevention/risk/infectious-
agents/hpv-vaccine-fact-
sheet?fbclid=IwAR3KSeHYE4WOLtkcsAAKYcMYJoDdUDg89LCnsN3K4
7BCVITCAfCOr6ibLrQ#q4
• https://www.netdoctor.co.uk/medicines/infection/a8654/cervarix-hpv-
vaccine/

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Thank you!

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