BIOLOGICAL E.

LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

MFR Effective Date:

Supersede No.: 400032.02

Production of Haemophilus Influenzae type b Crude
PRP at 600L Batch size in fermentation
Purification of Hib crude PRP at 16.0L batch size
Production of Hib (PRP - TTd) Conjugate at 20 gram
batch size with TT detoxified with Lysine (CPTT) with
Batch Size:
increased CNBr concentration.
Production of Hib (PRP - TTd) Conjugate in Tris buffer
at 5 gram batch size with BPTT
Production of Hib (PRP - TTd) Conjugate in Tris buffer
at 20 gram batch size with BPTT.
Customer Name: NA
Change Control No.: xxxx/xx.01.13
 To include Grade-B area in the process.
 To include new sterile filtration container closure
system.
 To perform sterile filtration activity in grade-B area
using new container closure system.
Reason for Changes:
 To update List of critical consumables to make inline
with change in sterile filtration container closure
system.
 To update List of equipments to make inline with
current area modification.

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

TABLE OF CONTENTS

S. No. Content Page No’s

1. Abbreviations 3-4

Precautions to be followed during the handling and

2. 5-7
processing

3. Bill of Materials 8-11

4. Calculations 11

5. Stages of Batch Manufacturing 12-56

List of Critical Control Points (CCPs)/Critical Quality

6. 57-64
Attributes (CQAs)

7. List of Major Equipments 65-67

8. List of Critical Process Consumables 68-69

9. Process Flow 70-74

10. Reference of Specifications Used 75-81

11. Master Formula Approval 82

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

1. ABBREVIATIONS:
 ADH : Adipic acid dihydrazide
 PRP : Polyribosyl ribitol phosphate
 AlPO4 : Aluminium phosphate
 QA : Quality Assurance
2.  BPR : Batch Production Record
 QC : Quality Control
 BP : British Pharmacopoeia
 q.s. : Quantity Sufficient
 BCA : Bicinchonic acid
 Qty : Quantity
 CMD : Cystine Magnesium Sulphate and Dextrose
 RPM : Rotation per minute
 CNBr : Cyanogen Bromide
 SOP : Standard Operating Procedure
 CIP : Clean In Place
 SS : Stainless Steel
 CPTT : Carrier protein Tetanus Toxoid
 SIP : Sterilization In Place
 CS-1
TTd :: Conjugate Suite 1
Tetanus Toxoid
 TFF
CTAB : Tangential Flow Filtration
Cetavlon trimethyl ammonium bromide
 UoM
DO : Units of Measurement
Dissolved Oxygen
 UFF
DOC : Ultra
Deoxyfiltration
Cholatesystem
 V/V
EDTA : Volume/Volume
Ethylene Diamine Tetra Acetic Acid disodium dihydrate
 WCB
EDC : Working
Ethylene cell bank
Carbodiimide
 WFI
ESIP : Water
Empty For Injection
Vessel Sterilization In Place
 W/V
GCR : Weight/volume
General Control Record
 GPC : Gel Permeation Chromatography
 HPLC : High Performance Liquid Chromatography
 HCl : Hydrochloric Acid
 ID No. : Identification number
 IPA : Iso propyl alcohol
 IPQC : In process Quality Control
 QC : Quality Control
 Kg : Kilo Gram
 kD : Kilo Daltons
 L : Litre
 mL : Milli Litre
Precautions to be followed during the handling and processing:

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

2.1 Instructions:

2.1.1 This Master formula is applicable to the following manufacturing process:

This Master formula is applicable to Manufacturing of Haemophilus
Influenzae type b vaccine bulk conjugate with three stages of manufacturing
process which consists manufacturing process of Crude PRP at 600L batch
size, Crude PRP Purification at 16L scale and PRP-TTd conjugation.
PRP-TTd conjugation methods are as follows:
 Haemophilus Influenzae type b vaccine bulk conjugate in PBS buffer
with TT detoxified with lysine (CPTT) and high CNBr concentration at 20g
batch size.
 Haemophilus Influenzae type b vaccine bulk conjugate in Tris buffer
with low CNBr concentration at 5g batch size.
 Haemophilus Influenzae type b vaccine bulk conjugate in Tris buffer
with BPTT and low CNBr concentration at 20g batch size.
2.1.2 Haemophilus Influenzae type b vaccine bulk conjugate in PBS buffer with
TT detoxified with lysine (CPTT) and high CNBr concentration at 20g
batch size consist of three stages of manufacturing process:
 Manufacturing process of Crude PRP at 600L batch size.
 Crude PRP Purification at 16L scale.
 PRP-TTd conjugation at 20 g batch size using detoxified TT with
Lysine(CPTT).
2.1.3 Haemophilus Influenzae type b vaccine bulk conjugate in Tris buffer with
low CNBr concentration at 5g batch size consist of three stages of
manufacturing process:
 Manufacturing process of Crude PRP at 600L batch size.
 Crude PRP Purification at 16L scale.
 PRP-TTd conjugation at 5 g batch size using BPTT.
2.1.4 Haemophilus Influenzae type b vaccine bulk conjugate in Tris buffer with
low CNBr concentration at 20g batch size consist of three stages of
manufacturing process
 Manufacturing process of Crude PRP at 600L batch size.
 Crude PRP Purification at 16L scale.
 PRP-TTd conjugation at 20g batch size using BPTT.
2.1.5 Production of each batch shall be carried out with approved BPR.
2.1.6 All the equipment shall be qualified before use and shall have approved
SOPs for their operation, cleaning and maintenance, wherever required.
2.1.7 Line clearance shall be carried out and recorded wherever applicable
during the production of a batch.

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

2.1.8 The usage of equipment, cleaning and operating parameters shall be

recorded in the respective equipment log books or general control records,
wherever applicable.
2.1.9 All the operations carried out during the production shall be recorded in
the BPR.
2.1.10 In-process and final product samples shall be collected as per the
requirements of this MFR and product specifications and submitted to QC.
Sampling activities and their testing results shall be recorded/ enclosed in
the BPR, wherever applicable.

2.2 Precautions:

 Haemophilus Influenzae type b is a live, pathogenic class 2

microorganisms, and extreme care should be taken during culture activity.
 Persons employed in production and quality control facilities should be
vaccinated against Haemophilus Influenzae type b.
 All live culture transfer activities should be done in closed containers or
in biosafety cabinet.
 Cell mass on harvesting should be promptly decontaminated by
appropriate method before its disposal.
 CNBr (Cyanogen Bromide) and EDC are toxic chemicals and should
be handled in fume hood after wearing protective garments and gloves.
Finally the wastes should be inactivated & disposed off properly.
 All autoclavable materials to be used in clean rooms should be pre-
sterilized in autoclave. Whenever required, QC approved, pre-sterilized
(sterile disposable material) pipettes, sampling tubes, bottles, etc. can be
used. Such material can be transferred to clean rooms after disinfecting
the exterior surface of bag/ pack.
 Material to be used should be dedicated & process specific to avoid
cross contamination.
 0.2 µm filters for sterile filtration are checked with pre-sterilization &
post filtration filter integrity.
 UFS/TFF systems shall be sanitized prior to start of operation and after
operation as per respective SOPs.
 Ethanol - Which is a flammable liquid and vapors may cause severe
eye irritation and respiratory tract irritation. Following safety measures are
to be followed while dispensing and handling of the solvent.
 Sufficient air and ventilation shall be provided while dispensing ethanol
in warehouse.
 Dispensing and handling of ethanol shall be done under the fume hood.
 Wear class B electrostatic garments while handling ethanol.

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

 Person should ware Neoprene gloves, goggles and nose mask.

 Nitrogen gas and fire extinguisher shall be provided during the
operation.
 Deoxy Cholate - Hazardous in case of eye contact may cause
irritation, of ingestion, of inhalation.
 Slightly hazardous in case of skin contact.
 Cetavalon –In case of eye contact, check for and remove any contact
lenses. In case of contact, immediately flush eyes with plenty of water for
at least 15 minutes. Cold water may be used. Get medical attention.
 In case of contact to skin, immediately flush skin with plenty of water.
Cover the irritated skin with an emollient. Remove contaminated Clothing
and shoes. Cold water may be used. Wash clothing before reuse.
Thoroughly clean shoes before reuse. Get medical attention.
 If inhaled, take a move to fresh air, If not breathing, give artificial
respiration. If breathing is difficult, give oxygen. Get medical attention.

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

3. BILL OF MATERIALS:
3.1 Raw materials required for the production of crude PRP at 600L
Fermentation:
Raw
Material Description
Raw material
(with Quantity/
S. No. Material UOM Specific
Pharmacopoeial Batch size
code ation
reference)
no.
1 099566 L-Glutamic acid 806.0 g 101688
Disodium hydrogen
2 099620 1550.0 g 101966
phosphate dehydrate
3 099560 Potassium Chloride 55.8 g 101626
4 099548 Sodium Chloride 3720.0 g 100617
5 099574 Ammonium Chloride 775.0 g 101684
6 099567 Soya-Peptone 300 g 101694
7 099559 Yeast extract 6240.0 g 101617
8 099558 Formaldehyde 3.0 L 101521
9 099572 Hemin 3.110 g 101702
10 099565 L-Cystine 9.330 g 101682
Magnesium sulphate
11 099533 373.2 g 101420
hepta hydrate
Dextrose (Glucose
12 099561 3.110 Kg 101628
mono hydrate)
13 099571 β-NAD 1.2 g 101700
14 070348 Hydrochloric acid 60 mL 100848
15 099550 Sodium hydroxide 800 g 100616

3.2 Raw materials required for the production of Purified PRP at 16.0 L batch
size:
Raw
Material Description
Raw material
(with Quantity/
S. No. Material UOM Specific
Pharmacopoeial Batch size
code ation
reference)
no.
Disodium Hydrogen
1 099620 84 g 101966
Phosphate
Sodium Dihydrogen
2 099538 24 g 101442
Phospahte
3 099548 Sodium Chloride 4510 g 100617
Hexadecyl trimethyl
4 099621 ammonium bromide 440 g 101698
(Cetrimide)
Sodium Deoxy
5 099568 128 g 101696
Cholate
6* 10058 Ethanol 150 L 101686

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BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

Sodium Acetate tri g 101704

7** 099573 4560
hydrate
Aluminium Chloride
8** 099557 8000 g 100622
hexa hydrate
Trisodium Phosphate
9** 099589 3300 g 100628
dodeca hydrate
10 099554 Tris 100 g 101511
11 099531 EDTA 12 g 101414
12** 099540 Sodium Carbonate 1500 g 101434

Note:
* Indicated materials shall be dispensed based on the Ethanol concentration
obtained as an assay. The value mentioned above varies from batch to batch.

** Indicated materials shall be dispensed based on the PRP concentration

obtained before AlPO4 gel addition. The value mentioned above varies from
batch to batch.

3.3 Raw materials required for the production of bulk conjugate (Purified PRP-
TTd conjugate) using PBS buffer at 20 g batch size with high CNBr
concentration and TT detoxified with Lysine (CPTT).:
Raw
Raw Material Description
Quantity/ material
S. No. Material (with Pharmacopoeial
Batch size UOM Specificati
code reference)
on no.
1 099537 Sodium bicarbonate 1019 g 101440
Sodium carbonate
2 099540 73 g 101434
anhydrous
3 09616 Adipic Acid dihydrazide 362 g 101964
4 099548 Sodium chloride 2800 g 100617
MES(2-
morpholinoethane
5 099618 310 g 101960
sulfonic acid hemi
sodium salt)
Disodium hydrogen
6 099620 2715 g 100623
phosphate di hydrate
Sodium dihydrogen
7 099538 1600 g 101442
phosphate dihydrate
8 099531 EDTA 470 g 101414
9 099615 Cyanogen bromide 160 mL 101955
10 099617 EDC 25 g 101962
Sodium Acetate
11 099573 60 g 101704
tri hydrate

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

3.4 Raw materials required for the production of bulk conjugate (Purified PRP-
TTd conjugate) using Tris buffer at 5 g batch size with Low CNBr and BPTT:
Raw
Raw Material Description
Quantity/ material
S. No. Material (with Pharmacopoeial
Batch size UOM Specificati
code reference)
on no.
1 099537 Sodium bicarbonate 255 g 101440
Sodium carbonate
2 099540 19 g 101434
anhydrous
3 099616 Adipic Acid dihydrazide 91 g 101964
4 099548 Sodium chloride 1173 g 100617
MES(2-
morpholinoethane
5 099618 207 g 101960
sulfonic acid hemi
sodium salt)
Disodium hydrogen
6 099620 1235 g 100623
phosphate di hydrate
Sodium dihydrogen
7 099538 651 g 101442
phosphate dihydrate
8 099531 EDTA 186 g 101414
9 099615 Cyanogen bromide 11 mL 101955
10 099617 EDC 4 g 101962
Sodium Acetate
11 099573 15 g 101704
tri hydrate
12 099554 Tris 90 g 101511

3.5 Raw materials required for the production of bulk conjugate (Purified PRP-
TTd conjugate) using Tris buffer at 20 g batch size with Low CNBr and BPTT:
Raw
Raw Material Description
Quantity/ material
S. No. Material (with Pharmacopoeial
Batch size UOM Specificati
code reference)
on no.
1 099537 Sodium bicarbonate 1019 g 101440
Sodium carbonate
2 099540 73 g 101434
anhydrous
3 099616 Adipic Acid dihydrazide 362 g 101964
4 099548 Sodium chloride 2199 g 100617
MES(2-
morpholinoethane
5 099618 310 g 101960
sulfonic acid hemi
sodium salt)
Disodium hydrogen
6 099620 2470 g 100623
phosphate di hydrate
Sodium dihydrogen
7 099538 1605 g 101442
phosphate dihydrate
8 099531 EDTA 376 g 101414

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

Raw
Raw Material Description
Quantity/ material
S. No. Material (with Pharmacopoeial
Batch size UOM Specificati
code reference)
on no.
9 099615 Cyanogen bromide 44 mL 101955
10 099617 EDC 16 g 101962
Sodium Acetate
11 099573 60 g 101704
tri hydrate
12 099554 Tris 135 g 101511

4. CALCULATION:
 NA

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

5. STAGES OF BATCH MANUFACTURING:

5.1 FERMENTATION
5.1.1 Process flow

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

5.1.2 Solutions for Fermentation

5.1.2.1 Preparation of Stock Solutions for Fermentation Activity:
 Pre-seed, Seed and Fermentor medium contains various chemicals. All
these chemicals are dissolved either separately or in combination prior to
use as stock solutions, sterilized either by filtration or by autoclaving and
mixed in appropriate concentrations to prepare medium for Pre-seed,
Seed and Fermentation stages.
5.1.2.2 Preparation of CMD stock solution:
 CMD is one of the components of the medium for seed as well as
fermentor. CMD is prepared by adding the chemicals, Magnesium
sulphate, Dextrose in WFI and L-cystine in 4N HCl as per SOP 102043.
Standard formula:
S. No. Material Name Standard Quantity for 1.0L
1. L-Cysteine 600 mg
2. Magnesium sulphate 24 g
3. Dextrose 200 g
4. 4M HCl 4 mL

5.1.2.3 Preparation of NAD stock Solution:

 NAD is one of the components of the medium for seed as well as
fermentor. It is dissolved in normal saline solution as per SOP 102040.
Standard formula:
S. No. Material Name Standard Quantity for 1.0L
1. NAD 400 mg
2. NaCl 580 mg

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

5.1.2.4 Preparation of Hemin stock Solution:

 Hemin is one of the components of the medium for seed as well as
fermentor. It is dissolved using sodium hydroxide solution as per
SOP 102041.
Standard formula:
S. No. Material Name Standard Quantity for 1.0L
1. Hemin 1000 mg
2. 0.1N NaOH 200 mL

5.1.2.5 Preparation of Yeast Extract stock solution:

 Yeast extract is one of the components of the medium for seed as well as
fermenter. It is dissolved in WFI and concentrated by using 30 kD
cassettes and collected the Permeate as per SOP 102042.
Standard formula:
S. No. Material Name Standard Quantity for 1.0 L
1. Yeast Extract 10.4 g

5.1.2.6 Preparation of 5.0M, 0.1N NaOH and 1M HCl:

 These solutions are prepared and used for pH adjustment in Fermentor
and sanitization of TFF system
Standard formula:
S. Standard Quantity for 1.0 L
Material Name
No. N/M Quantity/Volume
1. NaOH 5M 200 g WFI qs to 1.0 L
2. HCl 4N 8.33 mL WFI qs to 1.0 L
3 NaOH 0.1 N 4g WFI qs to 1.0 L

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

5.1.2.7 Preparation of Production solutions:

 Most of the solutions are freshly prepared for the Pre-seed, Seed and
Fermentation and sterilized either by filtration or by autoclaving and mixed
in appropriate concentrations to make medium for Pre-seed, Seed and
Fermentation.
5.1.2.8 Preparation of Basal salt medium for seed Fermentor:
 Basal salt media is used for cultivation of culture. Basal salt media is
sterilized in-situ in fermentor for 20 minutes at 121.5˚C.After sterilization
Yeast extract, Hemin, NAD and CMD stock solution are added into
fermentor aseptically.
Standard formula:
Standard Quantity
S. No. Material Name
for 20L
1. L-Glutamic acid 26.0g
2. Sodium chloride 120.0g
3. Di sodium hydrogen phosphate dihydrate 50.0g
4. Potassium chloride 1.8g
5. Ammonium chloride 25.0g
6. Soya Peptone 300.0g
After In-situ sterilization
7. Yeast extract stock solution 340mL
8. Hemin stock solution 100mL
9. CMD stock solution 500mL
10. NAD stock solution 100mL

5.1.2.9 Preparation of Yeast Extract solution:

 Yeast extract is one of the components of the basal medium for production
fermentor. It is dissolved in WFI and concentrated by using 30 kD
cassettes and collect the Permeate.
Standard formula:
S. No. Material Name Standard Quantity for 1.0 L
1. Yeast Extract 120

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

5.1.3 Preparation of fermentation medium:

 Basal salt media is used for cultivation of culture. Basal salt media is
sterilized in-situ in fermentor for 20 minutes at 121.5˚C.After sterilization
Yeast extract, Hemin, NAD and CMD stock solution are added into
fermentor aseptically.
Standard formula:
S. No. Material Name Qty for 600 L batch
1 L- Glutamic Acid 780 g
2 Di sodium Hydrogen Phosphate Di hydrate 1500 g
3 Potassium Chloride 54 g
4 Sodium Chloride 3600 g
5 Ammonium Chloride 750 g
6 Yeast extract solution 6240 g ( 52L)
7 5M NaOH q.s to adjust the pH 7.5±1
After In-situ sterilization
8 CMD 15000 mL
9 NAD 3000 mL
10 Hemin 3000

5.1.3.1 Preparation of PBS:

 PBS is used for diafiltration of Crude PRP. Raw materials are dissolved in
WFI and filtered using 0.22μ filter.
Standard formula
S. No. Material Name Qty for 1 L
1 Disodium hydrogen phosphate dihydrate 1.4 g
2 Sodium dihydrogen phosphate 0.35 g
3 Sodium chloride 9g
4 WFI q.s to 1.0L

5.1.4. Production details of MCB and WCB:

5.1.4.1 WCB of Haemophilus Influenzae type b

 From the master seed lot, working seed lots are prepared and stored at -
80°C in frozen state in secured separated deep freezers under the custody

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

of Production and Quality assurance. A culture of the working seed lot has
the same characteristics as that of the master seed lot and is confirmed by
tests for identity, purity and suitability for PRP production. A GCR is
maintained for working seed for reconciliation by production and cross
verified by QA.
 In order to set up a seed lot system during large scale production it’s
needed to produce enough MCB and WCB. That needed one time. After
release of the WCB the first large scale batch can be taken.
 For the production of MCB and WCB the same procedure can be followed.
 During the production of MCB and WCB the purity of the culture should be
checked regularly. After the harvesting of the MCB or WCB, pH should be
6.2-6.8 and OD590 should be 0.6-1.3 and a doubling time of about 1 hour,
the proper volume of 87% glycerol solution should be added.
 The solution should be homogenized, dispersed and frozen at <-80°C.
 GCRs shall be maintained for Cell Bank reconciliation.
5.1.5 Preparation of Pre seed , Seed and Fermentation Activities:
5.1.5.1 Pre-seed:
 Pre-Seed medium is prepared from the stock solutions as per SOP in a
suitable shake flask.
 Issue one working cell bank vial of Haemophilus influenzae type b
(760705) as per SOP 100457.
 Wipe out the WCB vial surface with 70% IPA haw one WCB vial, contains
8-10 ml of culture, inoculate in to the shake flask.
 Preseed growth conditions:
Temperature : 35˚C ± 2˚C.
pH : 5.8 to 6.8
Agitation : 200 RPM
Duration : 8 – 12 hours
OD590 : >1
 Preseed is harvested when OD590 is ≥ 1.0. Confirm culture purity by Gram
staining.
Acceptance: Gram stain: No evidence of contamination and should be
Gram negative coccobacilli.

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BIOLOGICS DIVISION
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

Preseed Culture is ready for inoculation of Seed fermentor.

5.1.5.2 Seed:
 To measure and control the Fermentation parameters, Fermentor consist
pH probe, DO probe and ensure cable connection with pH electrode to pH
transmitter & DO2 electrode to DO2 transmitter then Calibrate the pH and
DO probes prior to start the Fermentation as per SOP 102075.
 Fermentor CIP is done before leak test as per SOP 101061.
 Fermentor Air filters checked for Pre integrity test as per SOP 101279.
 Fermentor leak test performed prior to charge the medium as per
SOP 101489.
 Transfer lines are CIP & SIP able and is sterilized (Steam flushing for 45
minutes) prior to Fermentor ESIP.
 ESIP of Fermentor is done as per SOP 101075.
 Seed medium is prepared from stock solutions as per SOP 102044 and
transfer to seed fermentor.
 Complete Hib Basal Medium is sterilized in-situ at 121.5˚C for 20 minutes
in 70L fermentor. After cooling the medium to 35±1°C aliquot the Complete
Hib medium.
 Preseed is inoculated to 70L fermentor through hypodermic needle and
cultivated till the OD590 reaches to >2. Once OD590 reaches more than 2
stop the fermentation and check for purity, If broth is pure. Seed culture
transferred through pre sterilized transfer line from 70L fermentor to
Production fermentor.
 Used flasks and material shall be decontaminated as per load pattern No.
01 in autoclave as per SOP 101071.
 Seed growth conditions:
a) Temperature : 35°C ± 1˚C.
b) pH : 7.0
c) Agitation : 100 - 400 RPM
d) DO : 30%
e) RPM : 100 – 400
5.1.5.3 Acceptance criteria:
OD590: >2

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

Gram stain: No evidence of contamination and should be Gram negative

coccobacilli.
 After seed fermentation seed transferred to 1200 L production fermentor
though transfer line.
 After completion of transfer 70 L fermentor decontaminated and Air filters
checked for post integrity test as per SOP 101279.
5.1.5.4 Fermentation:
 To measure and control the Fermentation parameters, Fermentor consist
pH probe, DO probe and ensure cable connection with pH electrode to pH
transmitter & DO2 electrode to DO2 transmitter then Calibrate the pH and
DO probes prior to start the Fermentation as per SOP 102075.
 Fermentor CIP is done before leak test as per SOP 101061.
 Fermentor Air filters checked for Pre integrity test as per SOP 101279.
 Fermentor leak test performed prior to charge the medium as per
SOP 101489.
 Transfer lines are CIP & SIP able and is sterilized (Steam flushing for
45 minutes) prior to Fermentor ESIP.
 ESIP of Fermentor is done as per SOP 101075.
 Fermentation of Hib is done in fermentor with 1200 L capacity and working
volume 600 L. Fermentor is in-situ sterilized at 121.5°C for 20 minutes with
Basal salt medium with Yeast extract solution. Supplements are added
using siphon set. The seed is used to inoculate the fermentor through
transfer line from 70 L seed fermentor under aseptic conditions.
Fermentation is performed at following conditions and operational data
monitored and controlled by SCADA provided.
a) Temperature : 35 ± 1.0°C.
b) Agitation : 100 - 250 RPM
c) DO set point : 30%
d) pH : 7.0
 OD590 is monitored on spectrophotometer. Stop the fermentation after
getting two similar consecutive OD’s (OD 590>2) at two different time
intervals and check for purity. If broth is pure, harvest the culture and

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

centrifuged with Westfalia centrifuge (Continuous centrifuge) and separate

the Supernatant. Perform the CIP and SIP of Westfalia as per
SOP 101812 and 101815. Operate the Westfalia separator as per
SOP 101811.Add 0.1% formaldehyde to the sup and incubate for
overnight at 2 - 8°C.
 After completion of the Harvesting perform the CIP and SIP of Westfalia as
per SOP 101812 &101815.
 Perform CIP of the fermentor and check the post integrity of filters as per
respective SOP’s.
 After completion of centrifugation add Formalin to cell pellet to make a final
concentration of 1% v/v (10 mL formalin per 1L of cell pellet) into the 50L
carboy containing cell pellet and inactivate for 3 hours and direct to kill
tank line.
 Add Formalin solution to supernatant of Westfalia to make a final
concentration of 0.25%v/v. (2.5 mL per Liter of Supernatant) and incubate
at least 10 hours by circulating chilled water through the jacket of the
vessel.
 After Incubation the supernatant is concentrated to 30-50 times of the total
supernatant volume and diafiltered with 7-10 volumes of PBS buffer using
100 kD cassettes.
 TFF system for diafiltration should be sanitized with 0.5N sodium
hydroxide solution prior and after use as per SOP 102059.
 After diafiltration, Crude PRP is dispensed into Nalgene bottles. Label the
containers with batch details and container No. in roman letters. Store
them at -20˚C temperature.
 Crude PRP can be stored at -20˚C for 3 years.

5.2 PURIFICATION

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

5.2.1 PROCESS FLOW

Crude PRP
PRP Content
IPQC Test

CTAB addition

45% Ethanol Precipitation

0.45 µm Filtration
(Centrifuged Sup.)

72% Ethanol Precipitation

0.5% DOC + 32% Ethanol

Precipitation

PRP Content AlPO4 gel addition

IPQC Test

70% Ethanol Precipitation

QC Tests:
Identity
Molecular size
Diafiltration and 0.22µ filtration distribution
Ribose
Phosphorus
Protein
Nucleic acid
Endotoxin content
Moisture content
Pure PRP stored at -200C

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

5.2.2 SOLUTIONS PREPARATION:

5.2.2.1 PBS Buffer:
 PBS is used for dilution of crude PRP during the process. Prepared
solution is filtered through 0.22μ filter to trap the particulates and stored at
room temperature.
Standard formula:
Standard Quantity
S. No. Material Name
for 1L
1 Disodium hydrogen phosphate Dihydrate 1.4 g
2 Sodium Dihydrogen phosphate Dihydrate 0.40 g
3 Sodium Chloride 9.0 g
4 WFI q.s. to 1000 mL

5.2.2.2 10% CTAB Solution:

 CTAB solution is used for precipitation of Crude PRP.
 Dissolve the required quantity of CTAB in WFI at 35-45ºC for 15 minutes
using water bath.
 Prepared solution is stored at room temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0L
Hexadecyl trimethyl ammonium
1 bromide (Cetrimide/ Cetavlon/ 100 g
CTAB)
2 WFI q.s. to 1000 mL

5.2.2.3 Preparation of 0.5 M Sodium chloride:

 0.5 M sodium chloride is used for dilution of CTAB pellet. Prepared
solution is filtered through 0.22μ filter to trap the particulates and stored at
room temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1 L
1 Sodium chloride 29.22 g
2 WFI q.s. to 1000 mL

5.2.2.4 8M Acetic Acid:

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 8M acetic acid is used for pH adjustment at various precipitation steps.

Solution is prepared and stored in room temperature.
Standard formula:
Standard Quantity for 100
S. No. Material Name
mL
1 Acetic acid 45.7 mL
2 WFI 54.3 mL

5.2.2.5 Preparation of 4M HCl

 4M hydrochloric acid is used for pH adjustment at DOC buffer stage.
Solution is prepared and stored in room temperature
Standard formula:
Standard Quantity for 100
S. No. Material Name
mL
1 Hydrochloric acid 33.9 mL
2 WFI 66.1 mL

5.2.2.6 Preparation of DOC buffer:

 DOC buffer is used in the process after 72% ethanol precipitation. Pellet
obtained after 72% ethanol centrifugation is dissolved in DOC buffer and
incubated for 2 hrs at room temperature.
Standard formula:
Standard Quantity for
S. No Material Name
1.0L
1 Tris 6.057g
2 EDTA 0.740 g
3 Sodium Deoxycholate 3.0 g
4. WFI q.s. to 1000mL

5.2.2.7 Preparation of 20mg/mL AlPO4 gel preparation:

 AlPO4 gel is used for effective reduction of the endotoxin content in the
PRP. The solution is prepared and stored at room temperature.
Standard formula:

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Standard Quantity for

S. No. Material Name
1.0L
1 Aluminium chloride hexa hydrate 176 g
Trisodium phosphate dodeca 281.6 g
2
hydrate
3. WFI q.s. to 1000mL

5.2.2.8 Preparation of 25% sodium carbonate:

 25% sodium carbonate solution is used for pH adjustment of AlPO 4 gel
and is stored at room temperature.
Standard formula:
Standard Quantity for
S. No Material Name
1.0L
1 Sodium carbonate 250g
2 WFI 1000mL

5.2.3 Purification steps:

5.2.3.1 CTAB addition:
 Take the crude PRP container from the -20ºC deep freezer and keep it for
thawing. After thawing is complete, centrifuge the crude material at 6000
RPM at 2-8 0C for 30 minutes. Adjust the Centrifuged volume to 16L with
PBS buffer. If the Crude PRP Conc. (mg/mL) is more than 5 mg/mL, dilute
the sample with cool PBS to get the final concentration 5 mg/mL as per
below calculation

Calculation for PBS(mL) to added to get crude concentration 5 mg/mL.

= Crude PRP Conc. (mg/mL) x Centrifuge Vol. PRP (mL) – Centrifuge Vol. PRP (mL)
5
 Dilute the centrifuged / diluted crude PRP crude PRP four times with PBS
buffer.
 Add 10% Cetavlon solution to diluted crude PRP to a final concentration of
0.65% w/v at room temperature as per below calculation.

Calculation for 10% w/v Cetavlon solution to be added to the crude PRP

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

10% w/v Cetavlon (Cetrimide) = Volume of Crude diluted PRP x 1000 x 0.65
solution to be added in mL 10

 After addition of CTAB, stir the solution for proper mixing and incubate at
2-80C for at least 2hrs to overnight.

5.2.3.2 45% ethanol precipitation:

 After incubation, centrifuge the material at 2-80C for 30 mins at 4000 RPM.
 After centrifugation collect the pellet and dissolve in 0.5M NaCl.
 After complete dissolution, add ethanol to the solution to a final
concentration of 45% (v/v) to precipitate the contaminants/impurities like
Nucleic acids and proteins. Use following formula for calculating the
volume of ethanol to be added.
 Carryout the Ethanol addition under Fume hood.

Volume of 45
dissolved pellet ( X Actual concentration of Ethanol by assay - = Ethanol in L
In Liters) 45
 After addition of ethanol incubate the solution at 2-80C for at least 2-4hrs.
5.2.3.3 0.45 micron Filtration.
 After incubation, centrifuge the above material at 6000 RPM at 2-8 0C for
30 minutes.
 Perform 0.45 micron filtration for Supernatant collected.
5.2.3.4 72% ethanol precipitation:
 Collect the filtered supernatant and add ethanol to a final concentration of
72% (v/v) to precipitate the PRP. Use following formula for calculating the
volume of ethanol to be added.
 Carryout the Ethanol addition under Fume hood.

Volume of 72 - 45
supernatant X Actual concentration of Ethanol by assay – 72 = Ethanol in L
( In Liters)

 After addition of ethanol incubate the solution in 2- 8 0C for at least 2 hrs to

overnight.

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

5.2.3.5 Pellet in DOC buffer dissolution:

 Distribute the precipitated PRP in centrifuge bottles and centrifuge the
material at 4000 RPM at 2-8 0C for 30 minutes.
 Discard the supernatant and dissolve the pellet in DOC buffer and make
up to the initial volume of crude PRP taken for purification with DOC
buffer.
 Stir the solution for 2 hrs at room temperature.
 Add 60 g of Sodium acetate per liter to the dissolved pellet to a final
concentration of 6% (w/v).
5.2.3.6 0.5% DOC addition and 32% ethanol precipitation:
 Add 5.0 g of Sodium Deoxycholate per liter of dissolved pellet to a final
concentration of 0.5% (w/v)
 Adjust the pH to 6.2 ± 0.1 using 8M Acetic Acid.
 Add Ethanol to the dissolved pellet to a final concentration of 32% (v/v) as
per below calculation.

Volume of X 32
dissolved pellet Actual concentration of Ethanol by assay – 32 = Ethanol in L
( In Liters)
 Carryout the ethanol addition under Fume hood.
 If required adjust the pH to 6.8±0.1 by using 8M acetic acid.
 After addition of ethanol, incubate the solution at 2- 8 0C for at least 2 hrs to
overnight.
5.2.3.7 AlPO4 gel precipitation:
 Equally distribute the above precipitated solution in centrifuge bottles and
centrifuge the material at 6000 RPM at 2-80C for 30 minutes.
 Collect the supernatant and discard the pellet.
 Filter the solution through 0.22µ PES filter, and collect one sample to test
the PRP concentration.
 After knowing the PRP concentration, add the 20mg/ml prepared
aluminium phosphate gel in ratio of 1:10 (if the total PRP is 1 gram,

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

10grams of AlPO4 gel shall be added to the 32% ethanol precipitated

supernatant).
 Use following formula to calculate the volume of AlPO 4 gel to be added to
the solution.
Volume of
gel to be = volume of 32%sup after filtration (mL) X PRP (mg/mL) X 8
added Actual concentration of the gel

 Adjust the pH to 7.4± 0.1 using 25% Sodium carbonate.

 Incubate the gel added solution at room temperature for at least 8 - 12 hrs.

Removal of AlPO4 gel:

 After incubation, equally distribute the gel added material in centrifuge
bottles and centrifuge at 6000 RPM at 2-80C for 30 minutes.
 Collect the supernatant and discard the pellet.
Ethanol concentration before 70% ethanol precipitation:
 Prior to addition of the 70% Ethanol precipitation, calculate the ethanol
percentage by given below.

(Volume of 32% sup) X (32) .

(Volume of 32% sup)+ (Volume of gel)

5.2.3.8 70% ethanol precipitation:

 Supernatant collected from the above stage is filtered using 0.22µ filter to
remove any traces.
 Add 60 g of Sodium acetate per liter to the supernatant to a final
concentration of 6 % (w/v).
 Add 70 g of Sodium Chloride per liter to the supernatant to a final
concentration of 7 % (w/v).
 Adjust the pH to 6.3 ± 0.1 using 8M Acetic Acid.
 Add Ethanol to the above supernatant to a final concentration of 70% v/v
as per below calculation.
70-gel sup ethanol concentration of
Volume of X 32% = Ethanol in L
Supernatant Actual concentration of Ethanol by assay – 70

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
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( In Liters)

 Carryout the Ethanol addition under Fume hood.

 Adjust the pH to 6.8 ± 0.1 using 8M Acetic Acid.
 Store the material at 2 – 8˚C for at least 2 to overnight incubation.
5.2.3.9 Dia-filtration with WFI:
 After incubation, centrifuge the material at 6000 RPM at 2-8 0C for at least
15 minutes.
 Discard the supernatant and collect the pellet.
 Dissolve the pellet in WFI.
 After complete dissolution, the solution is diafiltered with 10 – 20 volumes
of WFI using 300 kDa cassettes.
 Collect the material after diafiltration and adjust the pH to 7.0±0.2 using
8M acetic acid or 0.1N sodium hydroxide if required.

5.2.3.10 Filtration of Purified PRP and Storage:

 Filter the purified PRP through 0.2 µ PES capsule filter.
 Collect the filtrate in depyrogenated glass bottle and collect sample for QC
analysis of Identity, Molecular size distribution, Ribose, Phosphorus,
Protein, Nucleic acid, Endotoxin content and Moisture content.
 Distribute the filtered purified PRP into sterile PP Nalgene bottles. Label
them with batch number, date of manufacturing, expiry date, storage
condition and done by.
 Store the bottle containing purified PRP in deep freezer at -20ºC.
Note: Assign the expiry date as per the specification SOP 102119.

5.3 CONJUGATION
 Manufacturing procedure for crude PRP (HICR) and purified PRP (HIPP)
is common for Hib bulk conjugate in PBS with increased CNBr

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

concentration and TT detoxified with Lysine (CPTT) at 20g batch size , Hib
bulk conjugate in Tris buffer with low CNBr concentration at 5g batch size
using BPTT and Hib bulk conjugate in Tris buffer with low CNBr
concentration at 20g batch size using BPTT .
 Starting materials for the Hib conjugation process are Hib Purified PRP ,
Bulk purified Tetanus Toxoid (BPTT) or Tetanus Toxoid detoxified with
Lysine (CPTT) as carrier protein for conjugation process.
 Hib Purified PRP which meets the specification as per specification SOP
102119 shall be used in the conjugation process. Purified PRP is stored at
or below -20ºC.
 Bulk purified Tetanus Toxoid (BPTT) which meets the specifications as per
SOP 100815 shall be used in the conjugation process as a carrier protein.
BPTT shall be stored at 2-8ºC.
 Carrier Protein Tetanus Toxoid (Detoxified with Lysine) which meets the
specifications as per SOP 103468 shall be used in the conjugation
process as a carrier protein. CPTT shall be stored at 2-8ºC.
 For manufacturing of Hib Bulk conjugate in PBS buffer with increased
CNBr concentration at 20g batch size, CPTT shall be used as starting
materials as protein source for conjugation.
 For manufacturing of Hib Bulk conjugate in Tris buffer with low CNBr
concentration at 5g batch size, BPTT shall be used as starting materials
as protein source for conjugation.
 For manufacturing of Hib Bulk conjugate in Tris buffer with low CNBr
concentration at 20g batch size, BPTT shall be used as starting materials
as protein source for conjugation.

5.3.1 Process flow of production of Hib Vaccine Bulk Conjugate in PBS buffer
using CPTT & high CNBr concentration
Purified PRP

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

PRP,
Concentrated PRP stock solution Mol. Size

NaHCO3/Na2CO3 Mol. Size

Buffer Alkaline Degradation

CNBr Activation Mol. Size

ADH + NaHCO3 Modification Mol. Size

Buffer

10X PBS Buffer & PRP, Degree of

PRP-ADH Concentration and Diafiltration Activation, Mol. Size
MES Buffer

Conc CPTT stock TTd

Con. CPTT in MES

PRP-ADH & TTd Conjugation Mol. Size
TT Conversion

NaH2PO4/Na2HPO4
PRP-TTd GPC Purification Mol. Size
Buffer + EDTA
PRP
TTd

PBS PRP-TTd Concentration and Diafiltration

with PBS

Pre Filtration (0.45 micron Filtration)

QC Tests:
Sterile Filtration (0.22 micron Filtration)
Identity
pH
PRP
Hib Conjugated Bulk TTd
PRP to Protein
Ratio
Free PRP
Mol. Size
Distribution
Sterility

5.3.2 Stock solutions and buffers preparation for conjugation

5.3.2.1 0.4 m Sodium carbonate-Sodium bicarbonate buffer, pH 10.5±0.1:
 0.4 m Sodium carbonate-Sodium bicarbonate buffer is required to degrade
the purified concentrated PRP during the process.

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 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0L
1. Sodium bicarbonate 5.04 g
2. Sodium Carbonate 36.04 g
q.s to 1000 mL
3. WFI
pH 10.5±0.1
5.3.2.2 ADH solution:
 1 M Sodium bicarbonate with ADH solution is required for modification of
degraded PRP.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0 L
1. Adipic Acid Dihydrazide 30.16 g
2. Sodium Bicarbonate 84.01 g
3. WFI q.s to 1000 mL
5.3.2.3 10X PBS Buffer pH-7.2±0.1:
 10XPBS Buffer pH-7.2±0.1 is required for diafiltration to remove free ADH
from modified PRP.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0 L
Disodium hydrogen phosphate
1. 13.8 g
di hydrate
Sodium dihydrogen
2. 3.616 g
phosphate dihydrate
3. Sodium chloride 88.0 g
q.s to 1000 mL
4. WFI
pH 7.2 ± 0.1

5.3.2.4 0.1M MES Buffer, pH 6.1±0.05:

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 0.1M MES Buffer, pH 6.1±0.05is required for buffer exchange of PRP-ADH

& carrier protein (TT).
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0L
MES (2 – morpholinoethane
1. 20.62 g
sulfonic acid hemi sodium salt)
2. Sodium chloride 29.22 g
q.s to 1000mL
3. WFI
pH 6.1 ± 0.05

5.3.2.5 Neutralization buffer , pH 8.0±0.1

 Neutralization buffer pH 8.0±0.1 is required for quenching the conjugation
reaction.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
S. No. Material Name Standard Quantity for 1.0L
Disodium hydrogen phosphate
1. 10.86 g
dihydrate
Sodium dihydrogen phosphate
2. 6.079 g
dihydrate
3. EDTA 1.86
q.s to 1000mL
3. WFI
pH 8.0 ± 0.1
5.3.2.6 10X PB with EDTA pH 7.0±0.1:
 10X PB with EDTA pH 7.0±0.1is required for GPC loading and purified Hib
conjugate fractions elution.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at room temperature.
 This is required to quench the conjugation reaction.
Standard formula
S. No. Material Name Standard Quantity for 1.0L
Disodium hydrogen phosphate
1. 10.86g
dihydrate

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
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S. No. Material Name Standard Quantity for 1.0L

Sodium dihydrogen phosphate
2. 6.079g
dihydrate
EDTA (ethylene diamine tetra
3. 1.86g
acetic acid disodium dihydrate)
q.s to 1000mL
4. WFI
pH 8.0 ± 0.1
5.3.2.7 PBS Buffer, pH 5.9±0.1:
 PBS Buffer, pH 5.9±0.1 is required for dia filtration (buffer exchange) of
purified Hib conjugate and removal of free PRP & TTd molecules.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0L
Disodium hydrogen phosphate
1. 0.234 g
dihydrate
Sodium dihydrohen phosphate
2. 1.326 g
dihydrate
3. Sodium Chloride 11.6 g
q.s to 1 L
4. Water For Injection
pH 5.9 ± 0.1
5.3.2.8 1.1M sodium acetate pH 5.5:
 1.1M sodium acetate pH 5.5 is required for sample dilution prior to loading
into HPLC.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
Standard Quantity for 1.0L
S. No. Material Name
N/M Quantity/ volume pH
1 Sodium acetate 1.1 M 149.68 g WFI qs to 1.0 L 5.5
5.3.2.9 Preparation of 5.0N ,0.1N, 0.5 N NaOH solution and 4N HCl solution:
 These solutions are prepared and used for pH adjustments and
sanitization of TFF and GPC system.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at room temperature.

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Standard formula:
Standard Quantity for 1.0L
S. No. Material Name
N/M Quantity/ volume pH
1 NaOH 5N 200 g WFI qs to 1.0 L NA
2 NaOH 0.5 N 20g WFI qs to 1.0 L NA
3 HCl 4N 8.33 mL WFI qs to 1.0 L NA
4 NaOH 0.1 N 4g WFI qs to 1.0 L NA

5.3.3 Process steps

5.3.3.1 Concentration of purified PRP:
 Native PRP is concentrated to ≥10 mg/mL using 100 kD cassettes under
stirring before starting the alkaline degradation. If purified PRP
concentration is already in this range, this step need not be performed.
 Concentration of PRP shall be prepared as stock and will be used with 10
days from date of preparation.
 Formula for PRP concentration is given below:
Volume of Conc. PRP (mL) =
Initial PRP Conc. (mg/mL) X Initial Volume of the PRP (mL)
10
 Formula for dilution of concentrated PRP to get target concentration of ≥10
mg/mL is given below:
Volume of diluent to be added (mL) =
PRP Conc.(mg/mL) x vol.of conc. PRP (mL)] - Conc. volume of PRP (mL)
10
5.3.3.2 Alkaline degradation and modification of Conc. Purified PRP:
 Concentrated native PRP is transferred to jacketed reaction vessel and
cooled to 2-4˚C under gentle stirring. On attaining the temperature 2-4˚C
equal volume of 0.4N sodium carbonate - bicarbonate buffer, pH 10.5 is
added. This reaction is performed at 2-8˚C till target PRP size reaches.
Samples are drawn at regular intervals and analyzed for MW by HPLC.
 This reaction is completed or stopped when molecular size is in the range
of 150 -250 kDa.
 CNBr (5M CNBr in Acetonitrile) solution is added to the degraded PRP
solution on the basis of 2.2 mL per gram of PRP. This reaction is
performed at 2-8˚C and stir for 10 minutes. Then pre-cooled sodium

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

bicarbonate with ADH solution is added immediately on the basis of

30grams per liter of Bicarbonate Buffer. This reaction is performed at 2-
8˚C for at least 14 hours. After Incubation aliquot 100 L of sample and
check the size by HPLC.
5.3.3.3 ADH removal, MES buffer exchange and concentration:
 After minimum 14 hrs incubation reaction mixture is concentrated to 20
times and diafiltered on 10 kD membrane using 20 volumes of 10X PBS
pH 7.2±0.1 to remove un-reacted ADH.
 On removal of un-reacted ADH, the PRP-AH is buffer exchanged with
0.1M MES buffer pH 6.1±0.05 and further concentrated to ~25mg/mL on
10kD membrane. The concentrated PRP-AH is stored at 2-8 ˚C and
analyzed for PRP contents and degree of activation and molecular size.
Degree of Activation value is for information only.
5.3.3.4 Preparation of Concentrated CPTT for conjugation:
 The carrier protein (TTd) is used for Hib conjugation process. Purified TT
produced using Lysine at detoxification stage with antigenic purity more
than 1500 Lf/mg of protein nitrogen is used for this process. The carrier
protein (TTd) is present in normal saline. It is buffer exchanged (diafiltered)
using 10 KDa against 5 volumes of 0.1 M MES pH 6.1±0.05 and
concentrated to ~25-30 mg/mL.
 Concentration of TTd shall be prepared as stock and will be used with 10
days from date of preparation.
 The concentrated TT is stored at 2-8˚C and analyzed for protein contents
by Lowry method.
 Acceptance Criteria : Protein 20-30mg/mL

5.3.3.5 Conjugation of PRP-AH with carrier protein TTd:

 The concentrated PRP-AH and TTd are mixed in jacketed reaction vessel
in the ratio (PRP: TT) 1 : 1.25 at 2-8˚C.
 EDC dissolved in 0.1M MES buffer pH 6.1±0.05 is added to the reaction
mixture. Amount of EDC added is equal amount of carrier protein.

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

 The reaction mixture is stirred at 2-4˚C and monitor the conjugation profile
at regular intervals on HPLC. Make up to total volume as per calculation
given below with 0.1M MES solution. Conjugation reaction will be stopped
at a minimum 85% of TT peak conversion is observed. After conjugation
reaction is over, it is quenched with addition of equal amount of phosphate
buffer with EDTA pH 8.0±0.1 (Neutralization buffer) and stir for 10minutes.
Sample is subjected to purification later.
 Formulas for conjugation is given below:
 Total reaction volume (mL) = 92.6 x amount of PRP (g)
 TTD volume required =1.25X amount of PRP(mg)
Concentration of TTd (mg/mL)
 Quantity of EDC to be added = Amount of TTd (mg)
 Volume of 0.1 MES solution required =amount of EDC (mg)/635
 Volume of 0.1M MES (mL) = Total reaction volume - (Vol. of PRP-ADH+
Vol. of TTd+ Vol. of EDC)
5.3.3.6 Purification of conjugate PRP-TTd:
 Purification of conjugate is done with GP Chromatography.
 Load conjugate PRP-TTd sample on to each column (not more than 5% to
the column volume) separately.
 For 20g scale, 4 GPC columns are used. The columns connections has to
be connected as follows;
Each AKTA pilot has a provision to keep two columns and need to connect
column as given in the below diagram.

AKTA pilot Column Inlet

Column Inlet

Column outlet

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Column outlet
 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

 Each column bed height should be more than 70cm.

 After injecting the sample to the column (Matrix: Sepharose CL 4B),
elution done using 10X PB with EDTA, pH 7.0±0.1 , fractions are collected
from the column based on the UV absorbance at 280nm and these are
analyzed for Molecular weight and ratio of PRP and TT.
 Pooled fractions shall be analyzed in IPQC for PRP-TTd ratio.
 Fractions with PRP-TTd ratio in the range of 0.30 - 0.55 and yield are
pooled based on the IPQC results obtained.
5.3.3.7 Diafiltrtaion of conjugate PRP-TTd:
 After pooling of all positive fractions concentrate twice to the initial volume
and perform Diafiltration of conjugate PRP-TTd using PBS buffer pH
5.90±0.1 up to 15-20 dia volumes.
5.3.3.8 Prefiltration of diafiltered bulk conjugate:
 Diafiltered bulk conjugate shall be filtered using 0.45 micron PES filter
prior to sterile filtration to reduce bioburden.
 Dia filtered bulk conjugate shall be stored at 2-8oC till sterile filtration.
5.3.3.9 Sterile Filtration of Bulk Conjugate:
 After diafiltration, the bulk conjugate is filtered through pre sterile 0.2µ PES
filter aseptically. Filter pre and post integrity shall be checked.
 Sterile filtration activity shall be performed in grade-B area sterile filtration
room (Room No: 6202) with new container closure system instead of using
sterile tubing welder and Thermoplastic tubing (C-Flex) for making aseptic
connections.
 Sterile filtration activity shall be performed under ceiling suspended LAFU
(ID No: 202917) located sterile filtration room (room No:6202) of grade-B
area.
 Sterile filtration bottle assembly shall be prepared with SS 2 way/PP 3 way
siphon and autoclaved as per validated loan pattern.

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

 After completion of sterile filtration, sterile filtered bulk container shall be

closed with sterilized cap under LAFU (ID No: 202917).
 Materials transfer to grade-B area shall be done through dynamic pass
box (ID No:202916) located in room No:6211 (Ultra filtration room-2).
 Samples shall be collected using pre sterilized pipettes using pipette
controller under LAFU (Id No:202917).
 Samples are to be sent to QC for analysis to analyze as per specification
SOP 102120.
 Label the bulk collected bottle with the batch no, date of manufacturing,
date of expiry, storage conditions and done by.
 Bulk antigen shall be stored at 2-80C till the batch release and further
usage.
 Assign the use shelf life as per SOP 102120.
Note: Formula provided for calculations in the MFR shall be used for each
container wherever multiple containers are used in the process.

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

5.4.1 Process flow of production of Hib Vaccine Bulk Conjugate in Tris at 5g

batch size using BPTT and Low CNBr
Purified PRP

PRP,
Concentrated PRP stock solution Mol. Size

NaHCO3/Na2CO3 Mol. Size

Buffer Alkaline Degradation

CNBr Activation Mol. Size

ADH + NaHCO3 Modification Mol. Size

Buffer

10X PBS Buffer & PRP, Degree of

PRP-ADH Concentration and Diafiltration Activation, Mol. Size
MES Buffer

Conc.TTd stock
solution TTd

Con.TTd in MES Mol. Size

PRP-ADH & TTd Conjugation
TT Conversion

NaH2PO4/Na2HPO4
PRP-TTd GPC Purification Mol. Size
Buffer + EDTA
PRP
TTd
Tris PRP-TTd Concentration and Diafiltration
with Tris

Pre Filtration (0.45 micron Filtration)

Sterile Filtration (0.22 micron Filtration) QC Tests:

Identity
pH
Hib Conjugated Bulk PRP
TTd
PRP to Protein
Ratio
Free PRP
Mol. Size
Distribution
Sterility

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

5.4.2 Stock solutions and buffers preparation for conjugation

5.4.2.1 0.4 m Sodium carbonate-Sodium bicarbonate buffer, pH 10.5±1:
 0.4 m Sodium carbonate-Sodium bicarbonate buffer is required to degrade
the purified concentrated PRP during the process.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0L
1. Sodium bicarbonate 5.04 g
2. Sodium Carbonate 36.04 g
q.s to 1000 mL
3. WFI
pH 10.5±0.1

5.4.2.2 ADH solution:

 1 M Sodium bicarbonate with ADH solution is required for modification of
degraded PRP.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0 L
1. Adipic Acid Dihydrazide 30.16 g
2. Sodium Bicarbonate 84.01 g
3. WFI q.s to 1000 mL

5.4.2.3 10X PBS Buffer pH-7.2±0.1:

 10X PBS Buffer pH-7.2±0.1 is required for diafiltration to remove free ADH
from modified PRP.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0 L
Disodium hydrogen phosphate
1. 13.8 g
di hydrate
Sodium dihydrogen
2. 3.616 g
phosphate dihydrate
3. Sodium chloride 88.0 g

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

S. No. Material Name Standard Quantity for 1.0 L

q.s to 1000 mL
4. WFI
pH 7.2 ± 0.1

5.4.2.4 0.1M MES Buffer, pH 6.1±0.05:

 0.1M MES Buffer, pH 6.1±0.05is required for buffer exchange of PRP-ADH
& carrier protein (TT).
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0L
MES (2 – morpholinoethane
1. 20.62 g
sulfonic acid hemi sodium salt)
2. Sodium chloride 29.22 g
q.s to 1000mL
3. WFI
pH 6.1 ± 0.05

5.4.2.5 Neutralization buffer , pH 8.0±0.1

 Neutralization buffer pH 8.0±0.1 is required for quenching the conjugation
reaction.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
S. No. Material Name Standard Quantity for 1.0L
Disodium hydrogen phosphate
1. 10.86 g
dihydrate
Sodium dihydrogen phosphate
2. 6.079 g
dihydrate
3. EDTA 1.86
q.s to 1000mL
3. WFI
pH 8.0 ± 0.1

5.4.2.6 10X PB with EDTA pH 7.0±0.1:

 10X PB with EDTA pH 7.0±0.1is required for GPC loading and purified Hib
conjugate fractions elution.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at room temperature.

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

 This is required to quench the conjugation reaction.

Standard formula
S. No. Material Name Standard Quantity for 1.0L
Disodium hydrogen phosphate
1. 10.86g
dihydrate
Sodium dihydrogen phosphate
2. 6.079g
dihydrate
EDTA (ethylene diamine tetra
3. 1.86g
acetic acid disodium dihydrate)
q.s to 1000mL
4. WFI
pH 8.0 ± 0.1

5.4.2.7 20mM TRIS Buffer, pH 7.0±0.1:

 20mM TRIS Buffer, pH 7.0±0.1is required for dia filtration (buffer
exchange) of purified Hib conjugate and removal of free PRP & TTd
molecules.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0L
Tris (tris (hydroxymethyl) amino
1. 2.242 g
methane)
q.s to 1000mL
2. Water For Injection
pH 7.0± 0.1
5.4.2.8 1.1M sodium acetate pH 5.5:
 1.1M sodium acetate pH 5.5 is required for sample dilution prior to loading
into HPLC.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
Standard Quantity for 1.0L
S. No. Material Name
N/M Quantity/ volume pH
1 Sodium acetate 1.1 M 149.68 g WFI qs to 1.0 L 5.5

5.4.2.9 Preparation of 5.0N ,0.1N, 0.5 N NaOH solution and 4N HCl solution:
 These solutions are prepared and used for pH adjustments and
sanitization of TFF and GPC system.

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at room temperature.
Standard formula:
Standard Quantity for 1.0L
S. No. Material Name
N/M Quantity/ volume pH
1 NaOH 5N 200 g WFI qs to 1.0 L NA
2 NaOH 0.5 N 20g WFI qs to 1.0 L NA
3 HCl 4N 8.33 mL WFI qs to 1.0 L NA
4 NaOH 0.1 N 4g WFI qs to 1.0 L NA

5.4.3 Process steps

5.4.3.1 Concentration of purified PRP:
 Native PRP is concentrated to ≥10 mg/mL using 100 kD cassettes under
stirring before starting the alkaline degradation. If purified PRP
concentration is already in this range, this step need not be performed.
 Concentration of PRP shall be prepared as stock and will be used with 10
days from date of preparation.
 Formula for PRP concentration is given below:
Volume of Conc. PRP (mL) =
Initial PRP Conc. (mg/mL) X Initial Volume of the PRP (mL)
10
 Formula for dilution of concentrated PRP to get target concentration of ≥10
mg/mL is given below:
Volume of diluent to be added (mL)=
PRP Conc.(mg/mL) x vol. of Conc. PRP (mL)] - Conc. volume of PRP (mL)
10
5.4.3.2 Alkaline degradation and modification of Conc. Purified PRP:
 Concentrated native PRP is transferred to jacketed reaction vessel and
cooled to 2-4˚C under gentle stirring. On attaining the temperature 2-4˚C
equal volume of 0.4N sodium carbonate - bicarbonate buffer, pH 10.5 is
added. This reaction is performed at 2-8˚C till target PRP size reaches.
Samples are drawn at regular intervals and analyzed for MW by HPLC.
 This reaction is completed or stopped when molecular size is in the range
of 150 -250 kDa.

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

 CNBr (5M CNBr in Acetonitrile) solution is added to the degraded PRP

solution on the basis of 2.2 mL per gram of PRP. This reaction is
performed at 2-8˚C and stir for 10 minutes. Then pre-cooled sodium
bicarbonate with ADH solution is added immediately on the basis of
30grams per liter of Bicarbonate Buffer. This reaction is performed at 2-
8˚C for at least 14 hours. After Incubation aliquot 100 L of sample and
check the size by HPLC.
5.4.3.3 ADH removal, MES buffer exchange and concentration:
 After minimum 14 hrs incubation reaction mixture is concentrated to 20
times and diafiltered on 10 kD membrane using 20 volumes of 10X PBS
pH 7.2±0.1 to remove un-reacted ADH.
 On removal of un-reacted ADH, the PRP-AH is buffer exchanged with
0.1M MES buffer pH 6.1±0.05 and further concentrated to ~25mg/mL on
10kD membrane. The concentrated PRP-AH is stored at 2-8 ˚C and
analyzed for PRP contents and degree of activation and molecular size.
Degree of Activation value is for information only.
5.4.3.4 Preparation of Concentrated TTd (from BPTT) for conjugation:
 Purified TT bulk antigen (BPTT) with antigenic purity more than 1500
Lf/mg of protein nitrogen is used for this process. Purified TT antigen is
present in normal saline. It is buffer exchanged (diafiltered) using 10 kD
against 5 volumes of 0.1 M MES pH 6.1±0.05 and concentrated to ~25-30
mg/mL.
 Concentration of TTd shall be prepared as stock and will be used with 10
days from date of preparation.
 The concentrated TT is stored at 2-8˚C and analyzed for protein contents
by Lowry method.
 Acceptance Criteria : Protein 20-30mg/mL
5.4.3.5 Conjugation of PRP-AH with carrier protein TTd:
 The concentrated PRP-AH and TTd are mixed in jacketed reaction vessel
in the ratio (PRP: TT) 1:0.8 at 2-8˚C.
 EDC dissolved in 0.1M MES buffer pH 6.1±0.05 is added to the reaction
mixture. Amount of EDC added is equal amount of carrier protein.

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

 The reaction mixture is stirred at 2-4˚C and monitor the conjugation profile
at regular intervals on HPLC. Make up to total volume as per calculation
given below with 0.1M MES solution. Conjugation reaction will be stopped
at a minimum 85% of TT peak conversion is observed. After conjugation
reaction is over, it is quenched with addition of equal amount of phosphate
buffer with EDTA pH 8.0±0.1 (Neutralization buffer) and stir for 10minutes.
Sample is subjected to purification later.
 Formulas for conjugation is given below:
 Total reaction volume (mL) = 92.6 x amount of PRP (g)
 TTD volume required =0.8 X amount of PRP(mg)
Concentration of TTd (mg/mL)
 Quantity of EDC to be added = Amount of TTd (mg)
 Volume of 0.1 MES solution required =amount of EDC (mg)/635
 Volume of 0.1M MES (mL) = Total reaction volume - (Vol. of PRP-ADH+
Vol. of TTd+ Vol. of EDC)
5.4.3.6 Purification of conjugate PRP-TTd:
 Purification of conjugate is done with GP Chromatography.
 Load conjugate PRP-TTd sample on to each column (not more than 5% to
the column volume) separately.
 For 5g scale, only 1 GPC column is used. The column connections has to
be connected as follows;
 Each AKTA pilot has a provision to keep two columns and need to connect
column, for the 5 g Scale only single column should be used.
Note: Since it is 5 g scale batch size, only one column shall be loaded for
the batch purification.

AKTA pilot Column Inlet

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

Column outlet

 Each column bed height should be more than 70cm.

 After injecting the sample to the column (Matrix: Sepharose CL 4B),
elution done using 10X PB with EDTA, pH 7.0±0.1 , fractions are collected
from the column based on the UV absorbance at 280nm and these are
analyzed for Molecular weight and ratio of PRP and TT.
 Pooled fractions shall be analyzed in IPQC for PRP-TTd ratio.
 Fractions with PRP-TTd ratio in the range of 0.30 - 0.55 and yield are
pooled based on the IPQC results obtained.
5.4.3.7 Diafiltrtaion of conjugate PRP-TTd:
 After pooling of all positive fractions concentrate twice to the initial volume
and perform Diafiltration of conjugate PRP-TTd using Tris as per
requirement.
 Diafiltration using TRIS buffer: Diafilter with 15-20 volumes of Tris buffer
pH 7.2 through 10 kDa cassettes.
5.4.3.8 Prefiltration of Diafiltered bulk conjugate:
 Diafiltered bulk conjugate shall be filtered using 0.45 micron PES filter
prior to sterile filtration to reduce bioburden.
 Dia filtered bulk conjugate shall be stored at 2-8oC till sterile filtration.
5.4.3.9 Sterile Filtration of Bulk Conjugate:
 After diafiltration, the bulk conjugate is filtered through pre sterile 0.2µ PES
filter aseptically. Filter pre and post integrity shall be checked.
 Sterile filtration activity shall be performed in grade-B area sterile filtration
room (Room No: 6202) with new container closure system instead of using

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 BIOLOGICAL E. LIMITED
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MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

sterile tubing welder and Thermoplastic tubing (C-Flex) for making aseptic
connections.
 Sterile filtration activity shall be performed under ceiling suspended LAFU
(ID No: 202917) located sterile filtration room (room No:6202) of grade-B
area.
Purified PRP
 Sterile filtration bottle assembly shall be prepared with SS 2 way/PP 3 way
siphon and autoclaved as per validated loan pattern. PRP,
Concentrated PRP stock solution Mol. Size
 After completion of sterile filtration, sterile filtered bulk container shall be
closed
NaHCO3/Na2CO with sterilized cap under LAFU (ID No: 202917).
3
Alkaline Degradation Mol. Size
Buffer
 Materials transfer to grade-B area shall be done through dynamic pass

CNBr
box (ID No:202916) locatedActivation
in room No:6211 (Ultra filtration room-2).
Mol. Size

 Samples shall be collected using pre sterilized pipettes using pipette

ADH + NaHCO3 controller Mol. Size
under LAFU (Id Modification
No:202917).
Buffer
 Samples are to be sent to QC for analysis to analyze as per specification
10X PBS Buffer & PRP, Degree of
MES Buffer SOP 102120. PRP-ADH Concentration and Diafiltration Activation, Mol. Size

 Label the bulk collected bottle with the batch no, date of manufacturing,
Conc.TTd stock
solution date of expiry, storage conditions and done by. TTd

 Bulk antigen shall be stored at 2-80C till the batch release and further
Con.TTd in MES
Mol. Size
usage. PRP-ADH & TTd Conjugation
TT Conversion
 Assign the use shelf life as per SOP 102120.
NaH2PO4/Na2HPO4
Note: Formula provided for calculations
PRP-TTd Mol.
in the MFR shall be
GPC Purification Size for each
used
Buffer + EDTA
PRP
container wherever multiple containers are used in the process.
TTd

Tris PRP-TTd Concentration and Diafiltration

with Tris

Pre Filtration (0.45 micron Filtration)

5.5.1 Process flow of production of Hib Vaccine Bulk Conjugate in Tris at 20 g

batch size using BPTTSterile
and Filtration
Low CNBr(0.22 micron Filtration) QC Tests:
Identity
pH
PRP
Hib Conjugated Bulk TTd
PRP to Protein
Ratio
Free PRP
Mol. Size
Distribution
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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

5.5.2 Stock solutions and buffers preparation for conjugation

5.5.2.1 0.4 m Sodium carbonate-Sodium bicarbonate buffer, pH 10.5±1
 0.4 m Sodium carbonate-Sodium bicarbonate buffer is required to degrade
the purified concentrated PRP during the process.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

Standard formula:
S. No. Material Name Standard Quantity for 1.0L
1. Sodium bicarbonate 5.04 g
2. Sodium Carbonate 36.04 g
q.s to 1000 mL
3. WFI
pH 10.5±0.1

5.5.2.2 ADH solution:

 1 M Sodium bicarbonate with ADH solution is required for modification of
degraded PRP.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0 L
1. Adipic Acid Dihydrazide 30.16 g
2. Sodium Bicarbonate 84.01 g
3. WFI q.s to 1000 mL

5.5.2.3 10X PBS Buffer pH-7.2±0.1

 10X PBS Buffer pH-7.2±0.1 is required for diafiltration to remove free ADH
from modified PRP.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0 L
Disodium hydrogen phosphate
1. 13.8 g
di hydrate
Sodium dihydrogen
2. 3.616 g
phosphate dihydrate
3. Sodium chloride 88.0 g
q.s to 1000 mL
4. WFI
pH 7.2 ± 0.1

5.5.2.4 0.1M MES Buffer, pH 6.1±0.05:

 0.1M MES Buffer, pH 6.1±0.05is required for buffer exchange of PRP-ADH
& carrier protein (TT).

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0L
MES (2 – morpholinoethane
1. 20.62 g
sulfonic acid hemi sodium salt)
2. Sodium chloride 29.22 g
q.s to 1000mL
3. WFI
pH 6.1 ± 0.05

5.5.2.5 Neutralization buffer , pH 8.0±0.1

 Neutralization buffer pH 8.0±0.1 is required for quenching the conjugation
reaction.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
S. No. Material Name Standard Quantity for 1.0L
Disodium hydrogen phosphate
1. 10.86 g
dihydrate
Sodium dihydrogen phosphate
2. 6.079 g
dihydrate
3. EDTA 1.86
q.s to 1000mL
3. WFI
pH 8.0 ± 0.1

5.5.2.6 10X PB with EDTA pH 7.0±0.1:

 10X PB with EDTA pH 7.0±0.1is required for GPC loading and purified Hib
conjugate fractions elution.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at room temperature.
 This is required to quench the conjugation reaction.
Standard formula
S. No. Material Name Standard Quantity for 1.0L
Disodium hydrogen phosphate
1. 10.86g
dihydrate
Sodium dihydrogen phosphate
2. 6.079g
dihydrate

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

S. No. Material Name Standard Quantity for 1.0L

EDTA (ethylene diamine tetra
3. 1.86g
acetic acid disodium dihydrate)
q.s to 1000mL
4. WFI
pH 8.0 ± 0.1

5.5.2.7 20mM TRIS Buffer, pH 7.0±0.1:

 20mM TRIS Buffer, pH 7.0±0.1is required for dia filtration (buffer
exchange) of purified Hib conjugate and removal of free PRP & TTd
molecules.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
S. No. Material Name Standard Quantity for 1.0L
Tris (tris (hydroxymethyl) amino
1. 2.242 g
methane)
q.s to 1000mL
2. Water For Injection
pH 7.0± 0.1
5.5.2.8 1.1M sodium acetate pH 5.5:
 1.1M sodium acetate pH 5.5 is required for sample dilution prior to loading
into HPLC.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at 2-80C temperature.
Standard formula:
Standard Quantity for 1.0L
S. No. Material Name
N/M Quantity/ volume pH
1 Sodium acetate 1.1 M 149.68 g WFI qs to 1.0 L 5.5

5.5.2.9 Preparation of 5.0N ,0.1N, 0.5 N NaOH solution and 4N HCl solution:
 These solutions are prepared and used for pH adjustments and
sanitization of TFF and GPC system.
 Prepared solution is filtered through 0.22μ filter to trap the particulates and
stored at room temperature.
Standard formula:

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

Standard Quantity for 1.0L

S. No. Material Name
N/M Quantity/ volume pH
1 NaOH 5N 200 g WFI qs to 1.0 L NA
2 NaOH 0.5 N 20g WFI qs to 1.0 L NA
3 HCl 4N 8.33 mL WFI qs to 1.0 L NA
4 NaOH 0.1 N 4g WFI qs to 1.0 L NA

5.5.3 Process steps

5.5.3.1 Concentration of purified PRP:
 Native PRP is concentrated to ≥10 mg/mL using 100 kD cassettes under
stirring before starting the alkaline degradation. If purified PRP
concentration is already in this range, this step need not be performed.
 Concentration of PRP shall be prepared as stock and will be used with 10
days from date of preparation.
 Formula for PRP concentration is given below:
Volume of Conc. PRP (mL) =
Initial PRP Conc. (mg/mL) X Initial Volume of the PRP (mL)
10
 Formula for dilution of concentrated PRP to get target concentration of ≥10
mg/mL is given below:
Volume of diluent to be added (mL)=
PRP Conc.(mg/mL) x vol. of conc. PRP (mL)] - Conc. volume of PRP (mL)
10
5.5.3.2 Alkaline degradation and modification of Conc. Purified PRP:
 Concentrated native PRP is transferred to jacketed reaction vessel and
cooled to 2-4˚C under gentle stirring. On attaining the temperature 2-4˚C
equal volume of 0.4N sodium carbonate - bicarbonate buffer, pH 10.5 is
added. This reaction is performed at 2-8˚C till target PRP size reaches.
Samples are drawn at regular intervals and analyzed for MW by HPLC.
 This reaction is completed or stopped when molecular size is in the range
of 150 -250 kDa.
 CNBr (5M CNBr in Acetonitrile) solution is added to the degraded PRP
solution on the basis of 2.2 mL per gram of PRP. This reaction is
performed at 2-8˚C and stir for 10 minutes. Then pre-cooled sodium
bicarbonate with ADH solution is added immediately on the basis of

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

30grams per liter of Bicarbonate Buffer. This reaction is performed at 2-

8˚C for at least 14 hours. After Incubation aliquot 100 L of sample and
check the size by HPLC.
5.5.3.3 ADH removal, MES buffer exchange and concentration:
 After minimum 14 hrs incubation reaction mixture is concentrated to 20
times and diafiltered on 10 kD membrane using 20 volumes of 10X PBS
pH 7.2±0.1 to remove un-reacted ADH.
 On removal of un-reacted ADH, the PRP-AH is buffer exchanged with
0.1M MES buffer pH 6.1±0.05 and further concentrated to ~25mg/mL on
10kD membrane. The concentrated PRP-AH is stored at 2-8 ˚C and
analyzed for PRP contents and degree of activation and molecular size.
Degree of Activation value is for information only.
5.5.3.4 Preparation of Concentrated TTd (from BPTT) for conjugation:
 purified TT bulk antigen (BPTT) with antigenic purity more than 1500 Lf/mg
of protein nitrogen is used for this process. Purified TT antigen is present
in normal saline. It is buffer exchanged (diafiltered) using 10 kD against 5
volumes of 0.1 M MES pH 6.1±0.05 and concentrated to ~25-30 mg/mL.
 Concentration of TTd shall be prepared as stock and will be used with 10
days from date of preparation.
 The concentrated TT is stored at 2-8˚C and analyzed for protein contents
by Lowry method.
 Acceptance Criteria : Protein 20-30mg/mL
5.5.3.5 Conjugation of PRP-AH with carrier protein TTd:
 The concentrated PRP-AH and TTd are mixed in jacketed reaction vessel
in the ratio (PRP: TT) 1:0.8 at 2-8˚C.
 EDC dissolved in 0.1M MES buffer pH 6.1±0.05 is added to the reaction
mixture. Amount of EDC added is equal amount of carrier protein.
 The reaction mixture is stirred at 2-4˚C and monitor the conjugation profile
at regular intervals on HPLC. Make up to total volume as per calculation
given below with 0.1M MES solution. Conjugation reaction will be stopped
at a minimum 85% of TT peak conversion is observed. After conjugation
reaction is over, it is quenched with addition of equal amount of phosphate

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

buffer with EDTA pH 8.0±0.1 (Neutralization buffer) and stir for 10minutes.
Sample is subjected to purification later.
 Formulas for conjugation is given below:
 Total reaction volume (mL) = 92.6 x amount of PRP (g)
 TTD volume required =0.8 X amount of PRP(mg)
Concentration of TTd (mg/mL)
 Quantity of EDC to be added = Amount of TTd (mg)
 Volume of 0.1 MES solution required =amount of EDC (mg)/635
 Volume of 0.1M MES (mL) = Total reaction volume - (Vol. of PRP-ADH+
Vol. of TTd+ Vol. of EDC)
5.5.3.6 Purification of conjugate PRP-TTd:
 Purification of conjugate is done with GP Chromatography.
 Load conjugate PRP-TTd sample on to each column (not more than 5%
to the column volume) separately.
 For 20g scale, 4 GPC columns are used. The columns connections has to
be connected as follows;
Each AKTA pilot has a provision to keep two columns and need to connect
column as given in the below diagram.

AKTA pilot Column Inlet

Column Inlet

Column outlet

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Column outlet
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
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 Each column bed height should be more than 70cm.

 After injecting the sample to the column (Matrix: Sepharose CL 4B),
elution done using 10X PB with EDTA, pH 7.0±0.1 , fractions are collected
from the column based on the UV absorbance at 280nm and these are
analyzed for Molecular weight and ratio of PRP and TT.
 Pooled fractions shall be analyzed in IPQC for PRP-TTd ratio.
 Fractions with PRP-TTd ratio in the range of 0.30 - 0.55 and yield are
pooled based on the IPQC results obtained.
5.5.3.7 Diafiltrtaion of conjugate PRP-TTd:
 After pooling of all positive fractions concentrate twice to the initial volume
and perform Diafiltration of conjugate PRP-TTd using Tris as per
requirement.
 Diafiltration using TRIS buffer: Diafilter with 15-20 volumes of Tris buffer
pH 7.2 through 10 kDa cassettes.
5.5.3.8 Prefiltration of Diafiltered bulk conjugate:
 Diafiltered bulk conjugate shall be filtered using 0.45 micron PES filter
prior to sterile filtration to reduce bioburden.
 Dia filtered bulk conjugate shall be stored at 2-8oC till sterile filtration.
5.5.3.9 Sterile Filtration of Bulk Conjugate:
 After diafiltration, the bulk conjugate is filtered through pre sterile 0.2µ PES
filter aseptically. Filter pre and post integrity shall be checked.
 Sterile filtration activity shall be performed in grade-B area sterile filtration
room (Room No: 6202) with new container closure system instead of using
sterile tubing welder and Thermoplastic tubing (C-Flex) for making aseptic
connections.

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

 Sterile filtration activity shall be performed under ceiling suspended LAFU

(ID No: 202917) located sterile filtration room (room No:6202) of grade-B
area.
 Sterile filtration bottle assembly shall be prepared with SS 2 way/PP 3 way
siphon and autoclaved as per validated loan pattern.
 After completion of sterile filtration, sterile filtered bulk container shall be
closed with sterilized cap under LAFU (ID No: 202917).
 Materials transfer to grade-B area shall be done through dynamic pass
box (ID No:202916) located in room No:6211 (Ultra filtration room-2).
 Samples shall be collected using pre sterilized pipettes using pipette
controller under LAFU (Id No:202917).
 Samples are to be sent to QC for analysis to analyze as per specification
SOP 102120.
 Label the bulk collected bottle with the batch no, date of manufacturing,
date of expiry, storage conditions and done by.
 Bulk antigen shall be stored at 2-80C till the batch release and further
usage.
 Assign the use shelf life as per SOP 102120.
Note: Formula provided for calculations in the MFR shall be used for each
container wherever multiple containers are used in the process.

6. LIST OF CRITICAL CONTROL POINTS (CCPs)/CRITICAL QUALITY

ATTRIBUTES (CQAs):
6.1 Critical Control Points (CCPs):
6.1.1 Critical Control Points (CCP): Hib Crude PRP at 600 L Batch size (HICR)
S.No. CCP Stage Justification

1 Volume of Pre-seed To have constant

Media inoculum percentage
2 Temperature Pre seed culture For optimal growth of
(35±2ºC) bacteria.
3 pH For optimal growth of

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

S.No. CCP Stage Justification

bacteria.
For better mixing of
4 media and increase
Rotation (200 RPM)
mass transfer coefficient
of oxygen.
To have optimal cell
5 Cell Density final density before
culture (OD>1) inoculating into seed
medium
6 Volume of Seed To have constant
Media inoculum percentage
7 Temperature For optimal growth of
(35±1°C) bacteria.
For optimal growth of
pH (7.0)
bacteria.
For better mixing of
8 media and increase
Rotation (200 RPM)
mass transfer coefficient
Seed Culture of oxygen.
To have optimal cell
9 Cell Density final density before
culture (OD>2) inoculating into
Production medium
Better aeration and
agitation with dissolved
10 Dissolved Oxygen
oxygen available for
(30%)
optimum growth of
microorganism
Fermentation To have constant
11 Volume of inoculum percentage
fermentation Media without nutrient depletion
throughout growth cycle
12 Temperature For optimal growth of
(35±1°C) bacteria.
13 For optimal growth of
pH (7.0)
bacteria.
For better mixing of
14 Agitation (100 - 250 media and increase
RPM) mass transfer coefficient
of oxygen.
Better aeration and
15 Dissolved Oxygen agitation available for
(30%) optimum growth and
release of PRP.
16 Cell Density final To have maximum

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

S.No. CCP Stage Justification

growth of with formation
culture (OD>2.0)
of PRP
To obtain clear
17 Flow rate at the inlet supernatant without cell
debris
To obtain clear
18 Centrifuge speed supernatant without cell
debris
Volume of Formalin Optimum concentration
19 added in supernatant of formalin for complete
Harvesting,diafiltration
(2.5 ml per liter if inactivation of bacterial
& concentration
supernatant) cells
20 Incubation Time Optimum time required
(NLT 10 hours) for complete inactivation
21 No of dia-volumes For removal of impurities
(atleast 7-10 times) less than 100kDa.
Volume of To have optimum
22 concentrated crude concentration of PRP for
PRP purification process

6.1.2 Critical Control Points (CCP): Purification of Hib Crude at 16L Batch size
(HIPP)
S.No. CCP Stage Justification

1 Concentration of PRP To decrease the input impurity

≤5mg/mL. load for the purificatin process
2 Final concentration of Optimum concentration required
Cetavelon (0.65 w/v) for precipitation of crude PRP.
For effective precipitation and to
3 Temperature during
prevent thermal degradation of
Incubation (2-8 ºC)
PRP
4 Optimal time required for
Incubation Time (NLT 2 hrs) CTAB
effective precipitation.
precipitation
For effective precipitation and to
5 Temperature during
prevent thermal degradation of
centrifugation (2-8 ºC)
PRP
Optimum centrifugation speed
6 Centrifugal speed (6000
(g force) to recover CTAB
rpm)
precipitate
7 Time of centrifugation (30 Optimum centrifugation time for
min) recovery of CTAB precipitate
45% ethanol Optimum pH for effective
8 pH (6.3±0.1) precipitation precipitation and to prevent
alkaline degradation of PRP.
9 Final concentration of Ethanol added for final

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

S.No. CCP Stage Justification

Ethanol (45% v/v) concentration (45 % v/v)

Optimum pH for effective
precipitation and to prevent
10 pH (6.8±0.1) alkaline degradation of PRP.
(pH increases after addition of
ethanol).
For effective precipitation and to
11 Temperature during
prevent thermal degradation of
precipitation (2-8 ºC)
PRP
12 Optimal time required for
Precipitation Time (2-4 hrs)
effective precipitation.
For effective precipitation and to
13 Temperature during
prevent thermal degradation of
centrifugation (2-8 ºC)
PRP
Optimum centrifugation speed
14 Centrifugal speed (6000
(g force) to recover 45%
rpm)
product in supernatant.
15 Time of centrifugation (30 Optimum time for Centrifugation
min) for to recover clear supernatant.
Optimum pH for effective
16 pH (6.3±0.1) precipitation and to prevent
alkaline degradation of PRP.
Ensuring ethanol concentration
17 Final concentration of
leads to maximum recovery of
Ethanol (72 %)
Pellets
Optimum pH for effective
precipitation and to prevent
18 pH (6.8±0.1) alkaline degradation of PRP.
(pH increases after addition of
ethanol).
19 Precipitation Time (2 hrs – 72% ethanol Optimal time required for
overnight) precipitation effective precipitation.
Optimum pH for effective
20 pH (7.0±0.1) diaggregation endotoxin
aggregates
For effective precipitation and to
21 Temperature during
prevent thermal degradation of
centrifugation (2-8 ºC)
PRP
Optimum centrifugation speed
22 Centrifugal speed (4000
(g force) for better recovery of
rpm)
pellet.
23 Time of centrifugation (30 Optimum time for Centrifugation
min) for to recover of pellet.
24 32% ethanol Optimum pH for effective
pH (6.2±0.1)
precipitation precipitation and to prevent

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

S.No. CCP Stage Justification

alkaline degradation of PRP.
(pH increases after addition of
ethanol)
Optimum ethanol concentration
25 Final concentration of leads to dissolve
Ethanol (32% v/v) polysaccharide without
impurities.
Optimum pH for effective
precipitation and to prevent
26 pH (6.8±0.1) alkaline degradation of PRP.
(pH increases after addition of
ethanol)
For effective precipitation and to
27 Temperature during
prevent thermal degradation of
precipitation (2-8 ºC)
PRP
For effective precipitation and to
28 Temperature during
prevent thermal degradation of
centrifugation (2-8 ºC)
PRP
Ideal centrifugation speed for
29 Centrifugal speed (6000
impurities to settle with clear
rpm)
32% ethanol supernatant.
30 Time of centrifugation (30 Optimum time for Centrifugation
min) for to recover clear supernatant.
Required for mass balance for
31 Volume of supernatant recovery and quantity of gel
required in gel addition step.
32 AlPO4 Al. Gel to be added calculating
PRP Value (For Information)
PRP value of Supernatant.
treatment
33 Optimum PRP to Gel ratio for
PRP AlPO4 gel ratio (1:10)
maximum removal of impurities.
Optimum pH for effective
34 pH (7.4±0.1) precipitation in presence of
Aluminium Phosphate gel.
Optimum temperature
35 Temperature during
Aluminium Phosphate gel
Incubation (RT)
precipitation.
Optimum time for incubation of
36 Incubation Time (8-12 hrs) Al. Phosphate gel for
precipitation.
For effective precipitation and to
37 Temperature during
prevent thermal degradation of
centrifugation (2-8 ºC)
PRP
38 Centrifugal speed (6000 Optimum centrifugation speed
rpm) ( g force) for gel treated 32%
supernatant recovery.

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

S.No. CCP Stage Justification

Optimum centrifugation time for
39 Time of centrifugation (30
maximum recovery of gel
min)
treated 32% supernatant.
Optimum pH for effective
precipitation and to prevent
40 pH (6.3±0.1) alkaline degradation of PRP.
(pH increases after addition of
ethanol)
Optimum ethanol concentration
41 Final concentration of
leads removal of impurities and
Ethanol (70% v/v)
maximum recovery of pellet.
Optimum pH for effective
precipitation and to prevent
42 pH (6.8±0.1) alkaline degradation of PRP.
(pH increases after addition of
70% ethanol
ethanol)
precipitation
For effective precipitation and to
43 Temperature during
prevent thermal degradation of
Incubation (2-8 ºC)
PRP
44 Incubation Time (2 hrs - Optimum incubation period for
Overnight) pellet precipitation.
For effective precipitation and to
45 Temperature during
prevent thermal degradation of
centrifugation (2-8 ºC)
PRP
46 Centrifugal speed (6000 Optimum centrifugation speed
rpm) (g force) for recovery of pellets.
47 Time of centrifugation (15 Optimum centrifugation time for
min) recovery of pellets.
48 Optimum pH to prevent alkaline
pH (7.0±0.1)
degradation of PRP
Maximum feed pressure For effective concentration and
49 during concentration diafiltration without loss of PRP.
/diafiltration (0.5-1.5 bar) 300kD
Diafiltration For removal of impurities less
50 No of dia-volumes (10-20 than 300kDa and traces of
volume of WFI) ethanol and aluminium
phosphate gel if any.
51 pH (7.0±0.2) Optimum pH for stable PRP.

52 0.22 micron To calculate the PRP recovery

Purified PRP Volume
filtration percentage

6.1.3 Critical Control Points (CCP): Hib Bulk conjugate HITT

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

S.No. CCP Stage Justification

Optimum TMP for performing
1 Maximum TMP during
PRP concentration of PRP
concentration
concentratio concentration without lossage
PRP Concentration (~10 n Optimum concentration before
2
mg/mL) degradation reaction

3 Degradation Optimum temperature to control

Temperature (2-4oC) degradation kinetics.
4 pH of Degradation buffer Optimum pH of buffer for alkaline
(10.5 ± 0.1) of PRP.
Alkaline
5 Rotation during To proper mixing during
Degradation
Degradation (180-220) degradation reaction.
Ensuring uniform molecular size
6 Target Molecular weight
of PRP used for conjugation and
(150-250 kDa)
to activate hydroxyl sites.
Volume of 5N CNBr Optimum concentration of CNBr
7 added (8 ml per gm of for cynalating the hydroxyl sites
PRP)
Volume of ADH- For effective formation of linker
8 carbonate buffer added molecules, PRP - AH
(3 volume of reaction
mixture) Activation
Optimum temperature for
9 Incubation Temperature effective degree of activation and
(2-80C) also to prevent further thermal
degradation of PRP-AH molecule.
10 Incubation time (at least Optimum time for incubation to
14 hours) form linker molecules.
Optimum TMP for performing
11 concentration/diafiltration of PRP-
TMP during Diafiltration
AH to remove unreacted
functional groups
No dia-volumes with Optimum volume of 10X PBS
12 10XPBS buffer (20 required for removal of unreacted
volumes) functional groups
No dia-volumes with PRP – AH To perform conjugation in
13 0.1MES buffer (5 Diafiltration 0.1MES buffer at pH6.1± 0.1
volumes) at 6.1± 0.1
To have concentration of
14 PRP Concentration 10mg/ml during conjugation
reaction
To have concentration of
15 Final concentration buffer
10mg/ml during conjugation
exchanged TT
reaction
16 Ratio of Activated Conjugation Optimum input PRP to TTd ratio
PRP:TT (1:1.25) for formation of stable conjugate

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S.No. CCP Stage Justification

with PRP – TTd ratio of 0.30 –
0.55
17 Temperature during Optimum temperature required
reaction (2-4˚C) for conjugation reaction.
18 TT conversion (to To confirm the conversion of TTd
conjugate) into TTd-PRP conjugate
Ensuring stable conjugate
GPLC
pH of the pooled fraction throughout the purification
19
purification process

Optimum diafiltration volume for

No. of dia-volumes (15-20
20 removal of residual unreacted
volumes)
functional groups
PRP – TTd
Optimum TMP for performing
Diafiltration
concentration/Diafiltration of
21 TMP during Diafiltration
PRP-TTd for effective buffer
exchange
To have constant concentration
22 Volume before filtration
prior to sterile filtration process
0.45 micron
To avoid leakage because of
Flow rate (250-500 filtration
23 high pressure drop across the
mL/min)
filter.
To have constant concentration
24 Volume before filtration
Sterile prior to sterile filtration process
To avoid leakage because of
Flow rate (250-500 filtration
25 high pressure drop across the
mL/min)
filter.

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

6.2 Critical Quality Attributes (CQAs):

S.No. CQA Stage Justification

1 For purity check of microorganism

Purity
at pre-seed stage.
2 Pre Seed To measure the growth of the
Optical Density
Culture organism in the medium.
3 For effective environment for
pH
growth of microorganism.
4 For purity check of microorganism
Purity
at pre-seed stage.
Seed culture
5 To measure the growth of the
Optical Density
organism in the medium.
6 To measure the growth of the
Optical Density
organism in the medium.
Fermenter
For verification for contamination
7 harvest
Purity and purity of microorganism in
medium
8 HICR after Concentration required to make up
PRP concentration
thawing volume before purification
9 Final Concentration of CTAB Optimum concentration required
Cetavelon Precipitation for precipitation of crude PRP
10 To measure required amount of gel
PRP Concentration
to be added for precipitation.
Critical, for addition of ethanol
11 AlPO4
Ethanol Assay during precipitation stages during
Treatment
purification of polysaccharide.
12 Critical for ensuring PRP to gel
PRP:AlPO4 gel ratio
ratio for precipitation
13 pH of Degradation Alkaline Critical to control the degradation
Buffer degradation reaction kinetics
Critical quality attribute for
14 Degree of activation Activation conjugation reaction and controlled
mol. size of conjugate
15 Critical parameter for effective
pH
conjugation kinetics.
16 Critical as lower temperature is
Temperature
required for conjugation.
To confirm the conversion of TTd
Conjugation
17 into TTd-PRP conjugate. Important
TT Peak Conversion
to avoid aggregate formation
during conjugation.
18 Critical for ensuring quality of PRP-
Molecular weight
TTd conjugate.

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

7 LIST OF MAJOR EQUIPMENTS:

7.1 Fermentation:
S. Equipment Manufacturer /
Model No. Capacity Use
No. Name Make
glassware
1. Autoclave Machine Fabrik NA NA
sterilization
2. Autoclave Machine Fabrik NA NA Decontamination
Spectronic
Spectro- Thermo Electron OD
3. 20+33318 NA
photometer Corporation Measurement
3
4. pH meter Polmon LP139S X 0 – 14 pH adjustment

5. Microscope Motic B1220ASC NA To check purity

NEC Culture
6. Incubator Newtronics NA
116PACS Incubation
Shaker New Brunswick Classic C- Culture
7. NA
Incubator Scientific 25KC Incubation
Laminar Air Aseptic
8. Fab Tech NA NA
Flow Unit processing
Media
9. Media Vessel Sartorius NA 800 L
Preparation
Seed
10. Fermentor Sartorius NA 70L
Fermentation
11. Fermentor Sartorius NA 1250L Fermentation
Westfalia GEA Westfalia AE1019 Culture
12. NA
Separator separator AG (CSC-6) Harvesting
Ultra Filtration YISMUF01
13. Pellicon NA Concentration
System L
YIICUF10L
Ultra Filtration Concentration &
14. Pellicon (JMBM018 NA
System Diafiltration
53)
ULT2540- Crude PRP
15. -20°C Freezers Thermo NA
5-V40 storage
DW- Crude PRP
40L626 (- storage &
16. -20°C Freezers Haier 460 L
40°C)Verti Purified PRP
cal low storage
17. Cold Room Carrier NA NA 20- 80C
Filter Integrity Filter Integrity
18. Pall NA NA
Tester Testing

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

7.2 PURIFICATION AND CONJUGATION:

S. Equipment Manufacturer /
Model No. Capacity Use
No. Name Make
glassware
1. Autoclave Machine Fabrik NA NA
sterilization
glassware
2. Autoclave Fedegari NA NA
sterilization
De-
Dry Heat
3. Machine Fabrik NA NA pyrogenation of
Sterilizer
glassware
Weighing CLWS1-
4. Sartorius 3 kg For weighing
Balance 3DC
Weighing
5. Sartorius BT 224S 220 g For weighing
Balance
Weighing
6. Metteler toledo IND 429 15 Kg For weighing
Balance
Weighing
7. Metteler toledo IND 465 60 Kg For weighing
Balance
Spectro-
8. Perklin Elmer Lambda 35 NA PRP estimation
photometer
Spectro- Agilent Cary PRP & protein
9. NA
photometer technologies 60UV-Vis estimation
Fischer Accumet
10. pH meter 0 – 14 pH adjustment
Scientific AB – 15
11. pH meter Thermo orion 2 star 0 – 14 pH adjustment

12. pH meter Thermo orion 3 star 0 – 14 pH adjustment

Millipore TFF
Concentration &
13. System (0.5 Millipore Pellicon NA
Diafiltration
m2)
Millipore TFF
Concentration &
14. System (0.1 Millipore Pellicon NA
Diafiltration
m2)
15. Centrifuges Beckman J – 20XP 6L Centrifugation
Chromatograp AKTA
16. GE NA Purification
hy system PILOT
17. HPLC System Waters 2695w NA Analytical
Haier DW-
-20°C Purified PRP
18. Haier 40L626 (- 460 L
Freezers storage
40°C)
To mix the
Magnetic Barnstead S1 33930- 1000
19. reaction
Stirrer Thermolyne 33 RPM
mixtures
20. Magnetic IKA MIDI MR 1 600 RPM To mix the

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

S. Equipment Manufacturer /
Model No. Capacity Use
No. Name Make
reaction
Stirrer DIGITAL mixtures and
chemicals
For
Laminar Air
21. Techno pak NA NA disinfectant/buff
Flow Unit
er filtration
0.45 micron
Laminar Air Dyna filters
22. NA NA filtration of bulk
Flow Unit private Ltd
conjugate
Laminar Air For 0.22µ
23. Fab Tech NA NA
Flow Unit filtration
Flame proof For ethanol
24. Citizen tech NA NA
hood addition
For Alkaline
25. Fume hood Citizen lab tech NA NA degradation &
Conjugation
Max
26. Water bath Julabo TW 20 IPQC
1000C
27. Cold Room Carrier NA NA 20- 80C
Storage of Bulk
28. Cold Room Blue star NA 20- 80C & in process
materials
Overhead Mixing of
29. Remi RQ 134L NA
stirrers solutions
Filter integrity
30. Integrity tester Millipore XIT4S0001 NA
testing
Sterile tube To make sterile
31. Terumo SCD IIB NA
welder connections
Packing of
32. Rotary Sealer Hawo HM850 NA materials for
sterilization
Ceiling Sterile filtration
Pharma
33. suspended CLAF5X3 NA of Hib bulk
engineers
LAFU conjugate
For materials
Dynamic pass Pharma
34. DPB-3X2.5 NA transfer to
box engineers
grade-B area
For Grade-B
Garment Dyna filters
35. NA NA garments
cubicle private Ltd
staging

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

8. LIST OF CRITICAL PROCESS CONSUMABLES:

Description of Vendor
S. Material MOC/
Critical Process Catalogue Use
No. Code Specification
Consumable No.
20 L Nalgene Poly
1. 70077 2250-0050, Product storage
carboy PP Propylene
Centrifuge Poly
2. 70347 352096 Sampling
tubes-15 mL Propylene
Centrifuge Poly
3. 70103 546041 Sampling
tubes-50 mL Propylene
Centrifuge Poly
4. 70348 REF352070 Sampling
tubes-50 mL Propylene
PP Bottles - 20 Poly
5. 70659 DCB20120 Product storage
L(Saint Gobain) Propylene
0.20 micron PDA020C02B Poly Ether
6. 70654 Filtration
filters 0.5 " AN1 Sulfone
0.20 micron PDA020J01A Poly Ether
7. 70655 Filtration
filters 10 " AN1 Sulfone
Sepharose CL-
8. 70427 170150-05 Agarose separation
4B matrix
Poly Tetra
Acro 37 vent Flouro
9. 70030 PN 4464 Sterile filtration
filter Ethylene
(PTFE)
Poly Tetra
Acrocap Vent
Flouro
10. filter 50mm 0.2 70031 PN 4251 Sterile filtration
Ethylene
micron
(PTFE)
Platinum Buffer
Silicon tubings
11. 70132 EK96403 cured preparations &
Size73
silicon filtration
Platinum Buffer
Silicon tubings 101408
12. WW-96410-35 cured preparations &
Size 35
silicon filtration
Platinum Buffer
Silicon tubings
13. 70131 96403-15 cured preparations &
Size 15
silicon filtration
Platinum Buffer
Silicon tubings 101407
14. WW-96410-17 cured preparations &
Size 17
silicon filtration
Platinum Buffer
Silicon tubings
15. 103821 WW-96410-24 cured preparations &
Size 24
silicon filtration
16. Schott duran 70011 21801245 Borosilicate Buffer
bottles with preparation,
screw caps, 100 sample

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

Description of Vendor
S. Material MOC/
Critical Process Catalogue Use
No. Code Specification
Consumable No.
ml collection
Schott duran
17. bottles with 70018 21801915 Borosilicate Product storage
screw caps 20L
Schott Duran
70011 21801245
bottles with Sample
18. 70012 21801365 Borosilicate collection
screw caps, 100
101406 21801175
ml &250 mL
Siphon set (3 Platinum
19. 70278 2132-1003 Sterile filtration
ports) cured silicone
C-flex &
20. EZ top cap 3port 103617 EZU20L-45-2 Platinum Sterile filtration
cured silicone
Container
closure for HITT
SS 316L with
21. SS 2 way siphon 151426 NA sterile bulk
GL-45 cap antigen during
sterile filtration
Buffer
Glass bottle O - Poly preparation,
22. 103747 10899 11
rings propylene sample
collection
Poly
23. Connectors (PP) 101610 ww-30612-15 Sterile filtration
propylene
Poly
24. Connectors (PP) 101609 ww-06365-22 Sterile filtration
propylene
Connector Poly Sterile filtration
25. straight, PP, 1/8 101609 EW-06365-22 propylene
inch
Connector Poly Sterile filtration
26. straight, PP, 1/4 103822 WW-96410-24 propylene
inch
Reducing Poly Sterile filtration
27. connector, PP 103823 SN 41518-36 propylene
1/4X1/8 inch
Poly Sterile filtration
28. PP connector 070849 EW-30612-59
propylene
0.45 micron filter 544306G0- Poly Ether
29. 70306 Filtration
capsule SS-V Sulfone
Centriguge Poly
30. 70423 363678 Centrifugation
Bottels propylene
0.45 micron filter Poly Ether
31. 70376 5442506G1 Filtration
catridge Sulfone
32. 0.2 micron PES 70655 PDA020J01A Poly ether Sterile filtration

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

Description of Vendor
S. Material MOC/
Critical Process Catalogue Use
No. Code Specification
Consumable No.
filter, size 10 "
AN1 sulphone
( Make: 3M)
Pipette sterile 70335 & 357550 & Poly
33. Sampling
50mL&100mL 70337 357600 propylene

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

9. PROCESS FLOW:
9.1 Fermentation:

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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

9.2 Purification:

Crude PRP
PRP Content
IPQC Test

CTAB addition

45% Ethanol Precipitation

0.45µm Filtration
(Centrifuged Sup.)

72% Ethanol Precipitation

0.5% DOC + 32% Ethanol

Precipitation

PRP Content AlPO4 gel addition

IPQC Test

70% Ethanol Precipitation

QC Tests:
Identity
Molecular size
Diafiltration and 0.22µ filtration distribution
Ribose
Phosphorus
Protein
Nucleic acid
Endotoxin content
Moisture content

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

9.3 Conjugation:
9.3.1 Process flow of production of Hib Vaccine Bulk Conjugate in PBS buffer
Purified PRP

PRP,
Concentrated PRP stock solution Mol. Size

NaHCO3/Na2CO3 Mol. Size

Buffer Alkaline Degradation

CNBr Activation Mol. Size

ADH + NaHCO3 Modification Mol. Size

Buffer

10X PBS Buffer & PRP, Degree of

PRP-ADH Concentration and Diafiltration Activation, Mol. Size
MES Buffer

Conc CPTT stock

TTd

Con. CPTT in MES

PRP-ADH & TTd Conjugation Mol. Size
TT Conversion

NaH2PO4/Na2HPO4
PRP-TTd GPC Purification Mol. Size
Buffer + EDTA
PRP
TTd

PBS PRP-TTd Concentration and Diafiltration

with PBS

Pre Filtration (0.45 micron Filtration)

Sterile Filtration (0.22 micron Filtration) QC Tests:

Identity
pH
PRP
Hib Conjugated Bulk TTd
PRP to Protein
Ratio
Free PRP
Mol. Size
Distribution
Sterility

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

9.3.2 Process flow of Bulk Conjugate in Tris at 5 g batch size

Purified PRP

PRP,
Concentrated PRP stock solution Mol. Size

NaHCO3/Na2CO3 Mol. Size

Buffer Alkaline Degradation

CNBr Activation Mol. Size

ADH + NaHCO3 Modification Mol. Size

Buffer

10X PBS Buffer & PRP, Degree of

PRP-ADH Concentration and Diafiltration Activation, Mol. Size
MES Buffer

Conc.TTd stock solution

TTd

Conc. TTd in MES

PRP-ADH & TTd Conjugation Mol. Size
TT Conversion

NaH2PO4/Na2HPO4
PRP-TTd GPC Purification Mol. Size
Buffer + EDTA
PRP
TTd

Tris PRP-TTd Concentration and Diafiltration

with Tris

Pre Filtration (0.45 micron Filtration)

Sterile Filtration (0.22 micron Filtration) QC Tests:

Identity
pH
PRP
Hib Conjugated Bulk TTd
PRP to Protein
Ratio
Free PRP
Mol. Size
Distribution
Sterility

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

9.3.3 Process flow of Bulk Conjugate in Tris at 20 g batch size

Purified
Purified PRP
PRP

PRP,
Concentrated PRP stock solution Mol. Size

NaHCO3/Na2CO3 Mol. Size

Buffer Alkaline Degradation

CNBr Activation Mol. Size

ADH + NaHCO3 Modification Mol. Size

Buffer

10X PBS Buffer & PRP, Degree of

PRP-ADH Concentration and Diafiltration Activation, Mol. Size
MES Buffer

Conc.TTd stock solution

TTd

Conc. TTd in MES

PRP-ADH & TTd Conjugation Mol. Size
TT Conversion

NaH2PO4/Na2HPO4
PRP-TTd GPC Purification Mol. Size
Buffer + EDTA
PRP
TTd

Tris PRP-TTd Concentration and Diafiltration

with Tris

Pre Filtration (0.45 micron Filtration)

Sterile Filtration (0.22 micron Filtration) QC Tests:

Identity
pH
PRP
Hib Conjugated Bulk TTd
PRP to Protein
Ratio
Free PRP
Mol. Size
Distribution
Sterility

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 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

10. REFERENCE OF SPECIFICATIONS USED:

10.1 In-Process, Intermediates Specification
10.1.1 Purified PRP
Specifications/
S. Ref. SOP
Stage Tests Acceptance
No. No.
criteria
After 32%
PRP testing by
1. ethanol For information 102338
Orcinol method
precipitation

10.1.2 Bulk conjugate

10.1.2.1 Concentrated PRP
Specifications/
S.
Stage Tests Acceptance Ref. SOP No.
No.
criteria
Concentrated PRP testing by
01 ≥ 10mg/mL 103471
PRP Orcinol method

10.1.2.2 Alkaline degradation

Specifications/
S.
Stage Tests Acceptance Ref. SOP No.
No.
criteria
Alkaline Molecular size
01 150-250 Kda 103471
degradation by HPLC

10.1.2.3 Concentrated TTd

Specifications/
S.
Stage Tests Acceptance Ref. SOP No.
No.
criteria
Protein
Concentrated
01 concentration by 25±5 mg/mL 103471
TTd
Lowry method

10.1.2.4 Conjugation
Specifications/
S.
Stage Tests Acceptance Ref. SOP No.
No.
criteria
Conjugate peak
01 Conjugation ≥85% 103471
shifting by HPLC

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

10.1.2.4 GPC fractions

Specifications/
S.
Stage Tests Acceptance Ref. SOP No.
No.
criteria
PRP testing by
GPC pooled Orcinol method & PRP to TTd ration
01 103471
fractions Protein estimation 0.30 to 0.55
by BCA method

10.2 Bulk antigen /Final bulk/Final lot specifications

10.2.1 Specification for Purified PRP as per SOP 102119

S.
Tests Specifications
No.
1 Identity Should pass the test
2 Molecular size distribution Not less than 400 kDa
3 Ribose For Information only
4 Phosphorus For Information only
Not more than 1% calculated with
5 Protein (Lowry)
reference to dry substance
Not more than 1% calculated with
6 Nucleic acid
reference to dry substance
Less than 10 IU per µg of
7 Endotoxin content
polysaccharide weight using LAL test
8 Moisture content To be used for correction of dry weight
10.2.2 Specification on modified PRP:
S.
Tests Specifications
No.
1 Degree of activation For Information only
2 Molecular size For Information only

10.2.3 Specification of TTd, to be used for the conjugation:

10.2.3.1 Specifications for Carrier Protein Tetanus Toxoid (CPTT) IH as per
SOP 103468
10.2.3.2 Specifications of Bulk Purified Tetanus Toxoid (BPTT) BP as per
SOP100815

S. Tests Specifications

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

No.
Not less than 1500 Lf per mg of protein
1 Purity
(non dialyzable) nitrogen.
2 Specific Toxicity Should pass the test

10.2.4 Specification of sterile filtered bulk conjugate (purified PRP – TTd

conjugate) as per SOP 102120:

Note 1:
* Hib Vaccine Bulk conjugate manufactured with PBS buffer shall be tested for total and free PRP
using SOP 103074.

* Hib Vaccine Bulk conjugate manufactured with Tris buffer shall be tested for total PRP using SOP
102112 and free PRP using SOP 102117.

Note 2:
Specific Toxicity test shall be performed on process validation batches as per protocol requirement
and stability batches as per schedule.

Note 3:
#
.Cyanide content shall be tested only in Hib vaccine bulk conjugate manufactured with increased
CNBr concentration.

10.3 Reference SOPs

10.3.1 List of SOPs available for Fermentation:
S. No. SOP No. SOP Title
1 101066 Aseptic Sampling from Product Storage Vessel
2 101075 Aseptic Sampling from 70L and 1200L Fermentors
3 101087 Gowning and Personnel flow in Diphtheria and Pertussis area
4 101465 Gram Staining
Specifications of Master and Working cell bank of
5 102028
Haemophilus influenzae type b
6 101583 Preparation of Normal Saline
7 101489 Procedure for Fermentor leak test
8 101811 Operation of Westfalia Separator
9 101074 Operation and Calibration of pH meter
10 101625 CIP and SIP of SS vessels
11 102041 Preparation of Hemin stock solution
12 100457 Cell banking
Preparation of Nicotinamide adenine di-nucleotide (NAD)
13 102040
stock solution
14 102043 Preparation of Cystine, MgSO4 & Dextrose (CMD) stock

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

S. No. SOP No. SOP Title

solution
15 102042 Preparation of yeast extract stock solution
16 102044 Preparation of complete Hib seed medium
17 101814 Preparation and Filtration of 70% IPA
18 102074 Estimation of PRP content in “crude PRP” samples
19 101812 CIP of Westfalia separator
20 101815 Sterilization In Place of Westfalia separator
21 101675 CIP and SIP and Operation of Media Transfer line
22 101061 Operation of CIP program in Sartorius 70L Fermentor
23 101059 Operation of ESIP program in Sartorius 1200L Fermentor
24 102064 Transfer of Basal salt medium to 70 L Seed Fermentor
25 101060 Operation of CIP program in Sartorius 1200L Fermentor
26 101059 Operation of ESIP program in Sartorius 70L Fermentor
27 101057 Operation of FSIP program in Sartorius 70L Fermentor
28 101056 Operation of FSIP program in Sartorius 1200L Fermentor
Operation of Fermentation program in Sartorius 1200L
29 101054
Fermentor
Operation of Fermentation program in Sartorius 70L
30 101055
Fermentor
31 101831 SIP of Transfer line from Fermentor to Westfalia separator
32 101071 Operation and cleaning of Autoclave
33 101279 Operation and cleaning of Filter integrity tester
34 102075 Handling & Calibration of Fermentor pH & DO probes
35 100053 Review of Executed BPR and Batch Release

10.3.2 List of SOPs available for Purification:

S. No. SOP No. SOP Title
1 101087 Gowning and Personnel flow in Diphtheria and Pertussis area
2 101875 Gowning and personnel flow in conjugation suite -1
Operation, calibration and cleaning of Sartorius 6.0kg
3 101068
weighing balance.
Operation, calibration and cleaning of Sartorius – BT 224S
4 102218
Weighing balance
5 102058 Operation of Ultra filtration System
6 102059 Sanitization of cassettes used in Ultra filtration System
7 101944 Operation, Cleaning and monitoring of cold room
8 101947 Operation and cleaning of Ceiling suspended Laminar Air

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

S. No. SOP No. SOP Title

Flow Unit
9 102098 Operation and cleaning of Autoclave.
10 101065 Operation and cleaning of Beckman coulter centrifuge
Operation and cleaning of Magnetic stirrer
11 102216
( Model MIDI MR 1 DIGITAL)
Operation and Calibration of L/S peristaltic pump
12 101919
( Model No 7523-70)
Operation and Calibration of L/S peristaltic pump
13 101920
( Model No 7523-60)
Operation and Calibration of I/P peristaltic pump
14 101921
( Model No 77410-10)
15 102225 Safety operation for Ethanol handling in Hib Production area.
Operation and cleaning of bench type flame proof fume hood
16 102428
(make: Citizen Labtec)
17 102224 Preparation of 8M Acetic acid solution
Testing of filter integrity using Millipore filter integrity test
18 101932
instrument (Model No – XIT4S0001)
Batch / Lot Numbering of intermediate Bulks and stock
19 102183
solutions.
Operation and cleaning of Watson Marlow peristaltic pump
20 102215
( Model No: 323E)
Operation , calibration and cleaning of Sartorius CLWS1-3dc
21 102217
weighing balance
Operation and cleaning of ultra low temperature deep
22 102371
freezer(-20˚C) (Make : REVCO)
23 100053 Review of Executed BPR and Batch Release
24 102541 Control of Hib intermediate bulks within the Hib production
25 102226 BPR for Hib purified PRP.
Specifications of Haemophilus Influenzae type b (Hib) purified
26 102119
polysaccharide (Poly ribosil ribitol phosphate).
Operation and cleaning of -200C deep freezer (Make: Haier,
27 103260
Model: DW-40L626)
Operation and Cleaning of top mount stirrer
28 103321
(Model No. RQ - 1341L, Make: Remi)

10.3.3 List of SOPs available for Conjugation:

S. No. SOP No. SOP Title

1. 102894 Gowning and Personnel Flow, Material transfer through

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

Material airlocks in Conjugation Suite III

Preparation of sodium carbonate – Sodium bi carbonate
2. 102326
solution
3. 102323 Preparation of ADH solution
4. 102328 Preparation of 1.1M Sodium Acetate of pH-5.5
5. 102319 Preparation of 10X PBS solution of pH-7.2
6. 102324 Preparation of 0.1M MES solution
7. 102332 Handling of CNBr
8. 102320 Preparation of Neutralization buffer of pH-8.0
9. 102318 Preparation of 10X PB with EDTA of pH-7.0 for GPC
Preparation of Sodium hydroxide solution in different
10. 102321
normality.
11. 102335 Preparation of 4M Hydrochloric acid solution
12. 102219 Operation and cleaning of thermo Orion pH meter
13. 100075 Storage of materials, cleaning and monitoring of cold rooms
14. 101922 Operation and cleaning of Magnetic stirrer
Operation, Verification and cleaning of Sartorius - BT 224S
15. 102218
Weighing Balance
Operation and Cleaning of Walk in Fume hood
16. 103486
(Make : Citizen Labtec)
17. 101927 Operation of Julabo recirculatory bath
18. 102059 Sanitization of cassettes used in Ultra filtration system
Operation and Calibration of L/S peristaltic pump
19. 101920
( Model No 7523-60)
Operation and Cleaning of Fedegari Autoclave in
20. 102888
Conjugation Suite-III
Operation and Calibration of L/S peristaltic pump (Model No
21. 101919
7523-70)
22. 102257 Operation and cleaning of Akta pilot system
Preparation of materials for Autoclaving and handling of
23. 102897
autoclaved materials
Specification of Haemophilus Influenzae b (Hib) vaccine bulk
24. 102120
conjugate.
Specifications of Haemophilus influenzae type b (Hib)
25. 102119 purified polysaccharide (polyribosyl ribitol
phosphate)
26. 102336 Calibration of HPLC and GPC columns
27. 102337 Estimation of Protein concentration by Lowry method
28. 102338 Estimation of Hib polysaccharide ( PRP) by Oricnol method
29. 102339 Estimation of Protein concentration by BCA
30. 102340 Estimation of molecular size by HPLC

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 BIOLOGICAL E. LIMITED
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Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

31. 101885 Operation of the SCD IIB sterile tubing welder

Operation and Cleaning of Ceiling Suspended Laminar Air
32. 102893
Flow Unit in Conjugation Suite-III
Cleaning and Sanitization of Clean Rooms and controlled
33. 102896
area in Conjugation Suite – III
Preparation of Filter and Siphon Assemblies in Conjugation
34. 102898
Suite-III
Operation and Calibration of pH meter (Make: Thermo orion,
35. 102934
Model: 3 star) in Conjugation Suite-III
Operation and cleaning of Magnetic Stirrer (Model MIDI MR
36. 102216
1 DIGITAL)
Operation and Cleaning of Filter Integrity Tester (Millipore,
37. 102892
Model No – XIT4S0001) in Conjugation Suite-III
38. 101071 Operation and Cleaning of Autoclave
Operation and Cleaning of Cold Room in Conjugation Suite-
39. 103384
III
Operation and Calibration of Lambda 35 UV
40. 102465
Spectrophotometer
Operation of Waters 2695 Alliance HPLC system with 2998
41. 102342
PDA detector, 2414 RI detector and Empower 2 software
BPR for Hib (Purified PRP - TTd) Conjugation at
42. 103359
20 grams scale in Tris buffer with low CNBr concentration
Specifications of Haemophilus influenzae type b (Hib)
43. 102119
purified polysaccharide (polyribosyl ribitol phosphate )
44. 103468 Specifications for Carrier Protein Tetanus Toxoid (CPTT) IH
45. 100815 Specifications of Bulk Purified Tetanus Toxoid (BPTT) BP
46. 103608 Concentration of purified PRP for Conjugation process
47. 103607 Concentration of BPTT/CPTT for Conjugation process
Operation and Cleaning of Ceiling Suspended
48. 103620 Laminar Air Flow Unit (Make: Pharma Engineers,
Model: CLAF 5’X3’) in Conjugation Suite-III
Operation and Cleaning of Dynamic Pass Box
49. 103621 (Make: Pharma Engineers, Model: DPB-3X2.5) in
Conjugation Suite-III
Operation and Cleaning of Garment cubicle (Make:
50. 103622
Dyna filters private Ltd) in Conjugation Suite-III
51. 103619 Procedure for area Fumigation in Conjugation Suite-III

MASTER FORMULA APPROVAL

Ref. No.: 102666.03/1 Page 81 of 82

 BIOLOGICAL E. LIMITED
BIOLOGICS DIVISION
MASTER FORMULA RECORD
Haemophilus influenzae type b (Hib) Vaccine Bulk Conjugate
MFR No: 400032.03

Name & Designation Department Signature Date

Prepared by

Reviewed by

Reviewed by

Approved by

Ref. No.: 102666.03/1 Page 82 of 82