Bio-Bac, Part 10

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PRODUCTION METHOD

This document describes the general operations that must be developed for the

production of BIO-BAC:

APPLICATION

This document is to be used at the manufacturing procedure of BIO-BAC to be

developed at industrial scale.

RESPONSIBILITIES

The representant of the Company can authorize the performance of manufacture of

BIO-BAC at the factory.

The representant of the factory is responsible of authorizing the manufacture at

his/her enterprise.

The representant of the Department of Production of the factory will supervise

the manufacture of BIO-BAC at his/her company.

EQUIPMENT, MATERIAL AND RAW MATERIAL

EQUIPMENT

-. Bacteriologic incubator for shaking cultures regulated at (36± 1)ºC.

-. Water baths with thermostat.

-. Crossed flow filtration equipment with filtration capacity of volumes of 40

to 800L.

-. Unidirectional filtration equipment with filtration capacity of volumes up to

100L.

-. Fermentators for the culture of microorganisms with capacity of 8L, and 800L.

MATERIAL

-. Glass flasks for shaking cultures with capacity of 2 to 4L.

-. Wide mouthed glass receptacles with capacity for 20L.

-. PVDF filtration membranes of 0.22m m.

-. Prescribing glass ampules of 2mL.

RAW MATERIAL

-. Bacterial strains: CFM-1, CFM-2, CFM-3, CFM-4, CFM-5,CFM-6,CFM-7 and CFM-8.

-. Culture media FCM.

-. Deionized water European Pharmacopeia (EP).

-. Water for injection (EP).

-. Bacteriologic quality NaCl.

-. Ultra pure Phenol.

-. Phormaldehide water solution 37%.

-. Anti-foam, Structol J 660.

SAFETY MEASURES

The safety measures to be applied are general measures for the staff that work

at industrial areas of biologic preparations following the GMP regulation since

the micro-organisms that will be processed are apathogenic.

For each production process of BIO-BAC, we will provide the 8 work strains and

the culture medium FCM, specific to be included in your fermentators as well.

The indicated volumes that follow are estimated, therefore they could suffer

changes during the standardization process.

Starting from above you must follow the following production protocol for each

of the 8 strains.

PROCEDURE

-. Primary propagation of the strains (PPS).

-. Preparation of the culture medium.

-. Dissolve 35g of culture medium CM-P by litter of deionized water.

-. Prepare 500mL of culture medium.

-. Sterilize the medium at a temperature of 121ºC for 15 minutes.

-. It is not necessary to adjust pH.

Inoculation

Inoculate the culture medium with the cellular suspension contained in vials

supplied identified as FCM-1,FCM-2,FCM-3, FCM-4, FCM-5, FCM-6, FCM-7 and FCM-8.

Culture parameters

-. Temperature (36 ± 1)ºC .

-. Incubator at constant shaking at 100 r.p.m.

-. Time 15 to 20 hours.

Control of the PPS

When propagation ends perform the following controls:

-. Purity control.

-. Control count of the Colony Forming Units (CFU)

-. Optic Density 620nm (OD620)

-. Identify the culture containers indicating what follows: The abbreviations

SPS and the date (year, month, day) following the abbrevations CHF. Ex:

PPS-97.09.17-CHF.

SECONDARY PROPAGATION OF THE STRAINS (SPS)

-. Preparation of the culture medium.

-. Dissolve 154 g of culture medium of FCM-P by litter of deionized water.

-. Prepare 8L of culture medium (the volume of the expansion cultures depends on

the capacity of the fermentators. Prepare a volumen of culture equivalent to 1%

of the volume of the MF), in this case, 8L is 1% of a fermentation of 800L.

-. Add Struktol J660 1mL/8L of medium, to control foam.

-. Adjust pH at 7 ± 2 with H3PO4 2M. Keep this pH during fermentation.

-. Sterylise the culture medium at a temperature of 121ºC for 15 minutes.

Inoculation

Transfer the cellular suspension (500 mL), obtained as result of the PPS to the

8L of culture medium.

Culture parameters

-. Temperature: (36 ± 1)ºC

-. Shaking: (500 ± 100) r.p.m.

-. Steryl air flow: (0,5-1)L/min.

-. Dissolved Oxygen:Initial (98 ± 2)%, during the culture >0%.

-. PH = 7 ± 0,2.

-. Time = 7 ± 2 hours.

Controls of the SPS

-. Purity control.

-. Control Count of CFU.

-. Wet weight matter OD620.

Identify the culture containers indicating what follows: The abbreviations SPS

and the date (year, month, day) following the abbrevations CHF. Ex:

SPS-97.09.17-CHF.

MAIN FERMENTATION (MF)

Preparation of the culture medium.

-. Dissolve 35 g of culture medium FCM by litter of deionized water.

-. Prepare 800L of culture medium (the volume of culture depends on the capacity

of the fermentator).

-. Add Struktol J660 100mL/800L of medium, to control foam.

-. Adjust pH to 7 ± 2 with H3PO4 2M. Keep this pH during fermentation.

-. Sterylise the culture medium to a temperature of 121ºC for 15 minutes.

Inoculation.

Transfer the cellular suspension (8L), obtained as result of the SPS, to the 800

L of culture medium

Culture Parameters.

-. Temperature: (36 ± 1)ºC

-. Shaking: (150-350) r.p.m.

-. Steril air flow: (0,5-1)L/min.

-. Dissolved Oxygen : Initial (98 ± 2)%, during the culture >0%.

-. PH= 7 ± 0,2

Time: we wish to reach the stationary growing phase and it is not critical to

keep the stationary phase for several hours (maximum: 4 hours). The culture time

will be fixed for each type of bacillus strain, during the standardization of

the processes, in relation with, consumption of substrates and OD.

Note: the foam can be a problem during fermentation

Controls of the MF

-. Culture controls during MF

-. Follow up of the culture by OD620

-. Control of the pH 7 ± 2

Final controls of the MF

-. Purity control

-. Control count of UFC

-. Wet weight matter

-. OD620

HARVEST

Transfer the cellular suspension to adequate recipients for their later

processing.

Identify the culture containers indicating the following: The abbreviations MF

and the date (year, month, day) in which it was obtained,followed by the

abbreviations CHF. Ex. MF-97.09.17-CHF

ACHIEVEMENT OF CONCENTRATED CELULAR SUSPENSION (CCS).

Filtration for concentration.

Use a filtratation equipment PROSTAK (or similar filtration equipment at crossed

flow) with PVDF membrane of 0,22m . Filter the 800L of celular suspension

obtained during the fermentation process. Obtain a concentration degree of up to

20 times more concentrated.

Diafiltration with interchange of physiological salty solution

Use a filtration equipment PROSTAK (or similar filtration equipment at crossed

flow) with PVDF membrane filter of 0,22m m, filter the concentrated celular

suspension obtained in 5.1. Use a volume of salty physiological solution

(0.9%m/v) of 150L.

Transfer the CCS to containers of 20L, 10L in each container.

Identify the containers of the culture indicating the following: the

abbreviations CCS and the date (year, month,day) in which it was obtained,

followed by the abbreviation CHF. Ex.: CCS-97.09.17-CHF.

ACHIEVEMENT OF A SUSPENSION OF LISATED CELLS BY FREEZE/HEAT

-. Put under freeze/heat process the CCS obtained in 5, which will present a

volume of approximately 40L

-. Put the containers with the CCS in refrigerator at 4ºC for 3 hours.

-. Move the containers to freezer at -40ºC and keep them in it for 18 hours.

-. Put the containers in a water bath at 65 ± 5ºC until total thaw.

-. Repeat the process described in (6.1 to 6.4), 4 consecutive times.

-. Perform a total protein content (Kjendal) after each of the 4 stages.

Although we propose 4 repetitions, these must be done for the count of proteins

during a pilot trial in order to determinate if later freeze/heat cycles would

produce a higher protein concentration.

ACHIEVEMENT OF THE ACTIVE INGREDIENT FREE FROM THE CELULAR DETRITUS (AI)

Purifying filtration: filtrated Active ingredient (FAI)

-. Use a filtration equipment PROSTAK (or similar filtration equipmente at

crossed flow) with PVDF membrane filter of 0,22m m, filter the suspension

obtained in 6.

-. In order to know the quantity of cellular detritus, take a sample at the end

of the filtration process, determine OD620 before and after centrifugating the

sample at 4000 r.p.m. for 15 min.The absorvance difference must be less than

0,1.

-. Identify the containers indicating the following: the abbreviations FAI and

the date (year, month, day) in which it was obtained, followed by the

abbreviations CHF. Ex: FAI-97.09.17-CHF.

Diafiltration with interchange of physiological salty solution: Purified Active

Ingredient (PAI)

-. Use a filtration equipment PROSTAK (or similar filtration equipment at

crossed flow) with PVDF membrane filter of 0,22m m. Filter the PAI obtained in

7.1. Use a volume of salty physiological solution (0.9%m/v) 10 times bigger than

the volume of the solution to be filtered (proportion 1:10).

-. Control the diafiltration by analysis of the total protein content in the

filtered (Kjendal).

-. Identify the containers indicating the following: The abbreviations PAI and

the date (year, month, day) in which it was obtained, followed by the

abbreviations CHF. Ex.: PAI-97.09.17-CHF.

Concentration of the product: Concentrated Active ingredient.(CAI)

-. Use a filtration equipment at crossed flow with membrane filter 5000Daltons

of diameter of nominal pore. Pellicon, Biomax 5k, Polysulfone, to obtain a 2x

concentration, which will represent an aproximate volume of 20L.

-. Determine the total amount of proteins (Kjendal).

-. Identify the containers indicating the following: the abbreviations of CAI

and the date (year, month, day) in which it was obtained, followed by the

abbreviations CHF. Ex.: CAI-97.09.17-CHF.

-. Adjust by dilution the protein concentration to obtain our standard

concentration.

Achievement of the steril active ingredient (SAI)

-. Filter the CAI using filtration equipment of PVDF membrane of 0.22m m of

pore.

-. Take samples in order to perform sterility and protein concentration tests

(Kjendal).

-. Identify the containers indicating the following: the abbreviations SAI and

the date (year, month, day) in which it was obtained, followed by the

abbreviations CHF. Ex.: SAI-97.09.17-CHF.

-. Store the SAI in cold chamber at 4ºC.

PREPARATION OF THE PRODUCT IN BULK (

Mix the SAI procedent from the 8 Bacillus strains FCM1, FCM2, FCM3, FCM4, FCM5,

FCM6, FCM7 and FCM8.

-. Use an adequate recipient and add an aproximate volume of 100L of water for

injection under EP.

-. Keeping at constant agitation, add phenol at a concentration of 0.2%(m/v) in

relation with the final volume.

-. Keeping at constant agitation, add phormaldehide solution at a concentration

of 0.02%(v/v) in relation with the final volume under EP.

-. Keeping at constant agitation add in proportion 1:1:1:1 the active ingredient

of the 4 strains.

-. Add water for injection, enough to complete an approximate volume of 200L and

keep in agitation (shaking) for 30min.

Steril filtration of the product in bulk

-. Filter the product in bulk using a filtration equipment with PVDF membrane of

0.22m m of pore.

-. Take samples to perform sterility and concentration of proteins tests

(Kjendal).

-. Identify the containers indicating the following: the abbreviation B and the

date (year, month, day), followed by the abbreviation CHF. Ex: B-97.09.17-CHF.

Keep the bulk product in refrigerator at 4ºC until following process.

Quality control of the Bulk. (See Annexe " Release Certificate " ).

FILLING PROCESS

Perform the filling process of the product, filling at a rate of 2mL by ampule,

under European Union Norms (EUN) for parentheral drugs.

Take samples for Quality Control. (See Annexe " Release Certificate " ).

REVISION

LABELING

PACKAGING

TECNICAL DEPARTMENT STAFF

Macario García Calvo. Mª del Mar Castellano Moreno.

Molecular Biotechnologist Msc. Molecular Biochemist Msc.

Greta Maluff . Mª Jesús Bermejo San Román.

Physician Microbiologist Msc. Physician MD.

Mª Angeles Vaqueriza Muñoz.

Biochemist Msc R+D.