FERMENTATION METHOD FOR

PRODUCING HYALURONIC
ACID: Streptococcus
zooepidemicus

Dr. Satheesh Kumar Gudi,

(satheesh.mcb@gmail.com)
 FERMENTATION METHOD FOR PRODUCING HYALURONIC ACID
(100 L Batch): USP & DSP

Unit process: USP Unit process: DSP

Ion Exchange Chromatography TFF: CPP’s
Brain heart infusion culture medium Clarification CPP’s (Strong Cation exchanger) Flow rate/flux
Inoculum, Seed culture CPP’s Cooling TMP
PCV calculation (18-25 %) Membrane 3 kDa
Stage I – LB medium (5 mL X 2) - ~ 6 h CMA’s
Stage II - LB medium (50 mL X 2) - ~ 6 h Discharge time • Streamline SP – 7L Resin
Stage III – HCDM medium (390 mL X 5) ~ 6 h • WFI
• 2M NaCl
• WFI
• 50 mM Sod.acetate (pH
5.5)
• 70L Loading
6% of glucose, • Wash
• Elution
0.5% yeast extract
1.0% of polypeptone
0.005% polyether Trypsinization
type antifoam Protein assay (120 g)/Trypsin
(12 mg)/16 hrs

Fermentation
CPP’s
Ion Exchange Chromatography
(Strong Cation exchanger)

CMA’s
• TOYOPEARL SP-650
• WFI, 2M NaCl, WFI
• 50 mM Sod.acetate (pH
5.5), 70L Loading, Wash
Hyaluronic acid can be isolated from the cultivated mixture as follows. The cells and the other insoluble components in • Elution

the cultivated mixture are first removed by filtration or centrifugal separation. Then, the proteins contained in the
resultant filtrate are removed by, for example, trichloroacetic acid or a mix
TFF: CPP’s
ture of chloroform and isoamyl alcohol, an adsorbent such as activated carbon or activated clay, or a Membrane 2 kDa, TMP, Flux
proteindecomposition enzyme such as pepsin, papain or pronase. The low molecular weight contaminants remain ing in
the cultivated mixture are removed by ultrafiltra tion, dialysis, organic solvent addition precipitation methods, a cationic
CQA’s: Sterile Filtration (DS)
surfactant, or an adsorption method using an ion exchange resin. Dr. Satheesh Kumar Gudi, 0.2 micron filtration, Bulk storage
(satheesh.mcb@gmail.com)
Facility Design: Concept note

• Bacterial fermentation suite design (Project plan)

• Autocad P&ID diagrams for process flow
• Area wise classification of equipment's (Culture area/Fermentation
area/Washing area/Solutions & Buffer preparation/Downstream process
area)
• Closed loop bioprocess layouts
• Utilities (Compressed air/Purified Water/Water for Injection/Pure
steam/Plant steam)
• Area classification and HVAC & AHUs
• As per GMP practices (A,B,C & D grades)
• Bill Of Materials (BOM)
• Instrumentation
• Automation
• Mechanical
• Equipements Installation & Qualification
• IQ,OQ,PQ
• Automation
• 21CFR Dr. Satheesh Kumar Gudi,
(satheesh.mcb@gmail.com)
HYALURONIC ACID Production Bioprocess: Growth &
Production kinetics

Dr. Satheesh Kumar Gudi,

(satheesh.mcb@gmail.com)
Facility Design: Concept note

• Bacterial fermentation suite design (Project plan)

• Autocad P&ID diagrams for process flow
• Area wise classification of equipment's (Culture area/Fermentation
area/Washing area/Solutions & Buffer preparation/Downstream process
area)
• Closed loop bioprocess layouts
• Utilities (Compressed air/Purified Water/Water for Injection/Pure
steam/Plant steam)
• Area classification and HVAC & AHUs
• As per GMP practices (A,B,C & D grades)
• Bill Of Materials (BOM)
• Instrumentation
• Automation
• Mechanical
• Equipements Installation & Qualification
• IQ,OQ,PQ
• Automation
• 21CFR Dr. Satheesh Kumar Gudi,
(satheesh.mcb@gmail.com)
Drug substance manufacturing process flow

Research Development Commercialization

Dr. Satheesh Kumar Gudi,

(satheesh.mcb@gmail.com)
Production (GMP) Projects

Bioprocess steps
(1) WCB/Flask-scale culture/Inoculum
25mL/500mL/2000mL
(2) Seed Fermenter/50L (2% inoculum)
(3) Production Fermenter/300L (5%
Inoculum)
(4) Cell Separation/Lysis
(5) Clarification/ TFF (300kDa)
(6) Purification
(7) Sterile Filtration

Dr. Satheesh Kumar Gudi,

(satheesh.mcb@gmail.com)
 Industrial Fermenter design

WFI
Clean Steam Plant Steam Compress
ed air
Utilities Chilled water SCADA/PLC cabinet

Fermentation
Vessel
Trouble shooting Fermenter preparation
• Pneumatic values • CIP
• PLC errors • Filters Integrity
• Leakages (PHT • PHT
failure) • ESIP
• Sensors

Drain Line Dr. Satheesh Kumar Gudi,

(satheesh.mcb@gmail.com)
 Dr. Satheesh Kumar Gudi,
(satheesh.mcb@gmail.com)