Chemistry Podcast

Showing posts with label Pharma Notes. Show all posts
Showing posts with label Pharma Notes. Show all posts

Tuesday, 19 January 2016

Media Filling Process

 Media Filling Process in Pharma Industry

Material issue as per FO (Formulation Order)
Material Verification
Media fill process
Material transfer into critical area (sterile lactose)
Liquid media preparation
Resealing of lactose bags
Check pH
Dispensing and blending with intervention simulation
Sterilize the m/c parts, rubber stoppers , dispensing aids, media garments and bin
m/c parts assembling ( filling) , bin , connection on the day of media filling
Media filling parts assembling and liquid media connection to filling m/c
Vial washing and sterilization

Media Checking for Growth

  •  Lactose and liquid media filling stoppering
  •  Sealing, crimping of aluminum seal ( sample for sterilize media for GPT, vials , rubber stoppers , compressed air sterility test / viable commit  monitoring)
  • Eternal vial washing m/c
  • Visual inspection m/c
  •  Online leak testing m/c
  •   Labeled tray with date / time and other details
  • Collect the vials in tray, transfer and handover to micro dept. for incubation through temp. controlled van (truck)

 Preparation of Media

·       Prepare 3% solution of media
·       30gm SCDM (soybean casein digest media) + 1000ml (qs) of wfi (Water For Injection) dissolve in glass carboy.
·       Collect the media sample for pH testing using pH meter and adjust the pH if require.
·       Labeled the media , date of preparation and exp date of media should be 7 days from the date of mfg (Manufacturing Date).
·       Plug the carboy with cotton plug and wrap with breathable cloth sterilize the carboy glass.
·       After sterilization unload the glass carboy in cooling zone and store under UAF on SS table (Stainless Steel Table).
·       Transfer the media carboy to vial filling room through the UAF mounted mobile trolley.
·       End of operation collect the media solution from each carboy and send to micro lab for GPT.
·       Incubate the media in micro lab for 7 days at 20C to 25C followed by 7 days at 30C to 35C.
·       Record the observation in microbiology validation green sheet.


          Growth Promotion Test, to insure that the liquid media used for the process simulation test is capable of microbial growth promotion.

Lactose Powder

                              Sterilized by gamma radiation method Lactose transfer in critical area
·       Gamma radiation sterilized powder of lactose => qc (quality Control) released => clean with lint free wipe
·       Outer carton and polybag removed in material transfer airlock
·       Pack sanitized and enter into pass box
·       Critical area
·       Lactose bags shall open inside the RABS (restricted access barrier system)

Dispensing Procedure

·       Lactose passes through mesh 36 over the ss-bin using the spoon
·       Load the bin on mixing column
·       Blend for 30 min
·       Unload the bin and transfer it into RABS
·       Remaining quantity also passed by same procedure
·       Quadruplet sampling carried out from 10 location (don’t take out sample only simulate)
·       Simulate pulled sample collection from 3 location (twice) for bulk analysis
·       Transfer the bulk containing lactose for vial filling operation

Assembling Vial Filling m/c Parts

·       Assembled the liquid media filling parts ( peristaltic pump with needles and silicon hose )
·       SS bin containing sterilize lactose
·       Upload it with lifting column
·       SS bin connect to powder hopper chute of vial filling m/c
·       Assembling the pre end post air flushing assembly
·       Insure that vials are flushed with sterile ( filtered ) compressed air during the filling process
·       30/40 ml vial – 50ml volume ( 250mg lactose) filled
·       100ml vial – 10ml volume ( 500mg lactose) filled
·       Minimum vial quantity to be put for incubation in each media fill operation shall not be less than 10,300 vials ( applicable for all pack sizes )
·       Record the collection time of media filled vials on each tray / boxes and affix the label

USE of Sterilize Lactose

·       Flow ability through the hopper and dosing wheel is similar to the product manufacture
·       It does not inhibit the growth of microbes and has growth promotion activity
·       Soluble in media and water
·       Sterilized by gamma radiation

Incubation Period

·       7 days ( bacterial growth ) – 20 to 25C
·       7 days ( viral growth ) – 30 to 35
·       Total 14 days incubation.
         1.  30/40ml vials 5500 to 6500 vials/hrs., lactose filled using single dosing mechanism      
            2. 100ml vials 2500 to 3500 double dosing wheel mechanism

Post Media-fill Activity

·       Clean and sanitize the equipment (used for media fill activity)
·       The final rinse sample (from product contact part) should be analyses for TOC
·       The TOC in the rinse water sample shall not exceed 500ppb
Tubular vial
Bromobutyle rubber stopper
Flip off

Media-fill Failure

·       In case of media fill failure respective procedure of OOS ( out of specification) in microbiological analysis , investigation , deviation management shall be followed
·       Characterization of microorganism up to species level shall be carried out to find out the sources of contamination
·       Environmental records for manufacturing and testing area for temp. RH , DP and non-viable air born particle counts
·       Microbiological monitoring, reports of manufacturing and testing area for settling plate air sampling, surface monitoring and personnel monitoring.

Sunday, 6 September 2015




                                Pathology is a branch of biological science which deals with the nature of disease, through a study of its causes, its processes and its effects, with associated structural and functional alteration. When a person becomes ill, his symptoms are due to disturbances in the normal functions of the affected cell of his body. Pathology deals with the study of these disturbed function, how they arise, how they and how they affects other cell system, pathology also consider the factor which return the function of the affected cell to normalcy.


                                              It is the application of laboratory techniques to find out the cause of disease. Clinical laboratory testing is regarded as one of the several important sources of clinical data essential for diagnosis, prognosis and treatment of disease. Clinical laboratory medicine involve all aspects of medicine ranging from field of biochemistry, microbiology, hematology, immunology, clinical microscopy, biophysics, cytogenetic to is topology etc..


                              Orders laboratory test in the order to arrive at a diagnosis when a patient approaches the laboratory with a requisition form, requesting pathological tests, the pathologist, with the help of other laboratory staff carries out the following duties –
Ø       Instructing the patient
Ø   Collecting the specimen
Ø   Preserving the specimen
Ø   Analyzing the specimen
Ø   Preparing the reports and
Ø       Dispatching of the typed and signed reports

The test reports are useful to the physician to-
Ø                  Establish a diagnosis
Ø       Confirm a clinical interpretation
Ø                   Monitor therapy
Ø       Establish prognosis
Ø                   Detect disease

The functional components of the clinical laboratory can be listed as follows-
Ø       Clinical pathology
Ø                   Hematology and Blood bank 
Ø       Clinical  biochemistry
Ø       Clinical microbiology and serology
Ø       Histology and cytology


Ø           The laboratory workers play an important role to find out the cause of disease by providing the physician the required laboratory test results.

Ø      The laboratory worker thus helps the patient to get better by providing accurate test finding to the physician.

Ø  The laboratory worker should not offer personal excuses for short-comings in the performance of duty.

Ø        Any error should be promptly reported to the superior without fear, so that they can be corrected.

Ø    Truth must always be told because a wrong result may lead to a patient’s death.

Ø    Proper use of equipments and reagents because equipment and chemicals coast money.

Ø      Many patients are not treated until their reports are kept ready. If these reports are delayed, patients cannot be treated early.

Ø  It is necessary to keep all reports ready in time (particularly the urgent reports).

Ø   In the course of laboratory tastings, the laboratory workers gain a lot of information about patients and their illnesses. The laboratory worker must regard this information as strictly confidential.

Ø  Only physician who requested the examinations should receive the patient’s reports.

Ø    Every laboratory worker must maintain high moral and professional standards of behavior.


The laboratory worker is surrounded by many dangers such as from-

Ø Handling of infectious material.

Ø Handling of broken glassware.

Ø Accidental spill of corrosive reagents.

Ø Swallowing of  corrosive reagents such as conc.H2SO4, HCl, NaOH etc.

Ø Swallowing of infectious specimen.

Ø Inhalation of poisonous fumes.

Ø Potential hazards in the form of inflammable chemicals and gas leakages.


Ø The use of rubber gloves while handling corrosive substances such as strong acid and alkalies  and also while handling poisonous chemicals such as potassium cyanide is essential.

Ø The uses of laboratory coats are meant to protect the wearer from chemical splashes and infectious material.

Ø Safety spectacles or goggles should be used while carrying out any procedures where there is risk to the eyes from reagent splashes.

Ø All chemical should be considered as potentially dangerous. Contact with skin and clothing should be avoided.

Ø A gas cylinder and gas taps should be handled with care. Accident can happen through ignorance and incorrect use.

Ø All body fluids such as blood, serum, plasma, urine, CSF, etc. should be handled with great care since they may be potential source of infections.

Ø Laboratory worker must know the meaning of safety signs for the following type of harmful substances.



                                         The laboratory glassware is usually manufactured from borosilicate glass. It is resistant to the action of chemicals with the exception of hydrofluoric acid.


Silica (SiO2)
Boric Oxide (B2O2)
Sodium oxide (Na2O)
Alluminium Oxide (Al2O3)


v Beakers:-

                  These have capacities from 5 to 5000 ml. they are generally in a square form which cylindrical and has a spout. These are used mainly for the preparation of solutions.

v Flasks:-

               These have capacities of 25 to 5000 ml. different types of flasks are used such as-
ü Conical flasks:- used for performing titrations.

ü Flat bottomed round flasks:- used for heating liquids.
ü Round bottomed flasks:- these can withstand higher temperature. They can be heated in a naked flame.

ü Volumetric flasks:- used to make final volume of reagent very accurately.

v Measuring cylinders:-

                                       They are available in 10 to 2000 ml capacities. They are used to measure quantity of the liquids.

v Burettes:-

                   These are used for measuring of variable quantities of liquids. These are used for titration.

v Condensers:-

                        Used mainly for distillation and for reflux operations.

v Funnels:-

                  These are available in comprehensive range for variety separations of-
ü Solids from liquids.
ü Liquids from liquids.
ü For pouring liquids, chemicals or solutions into a container.

v Syringes :-

                   These are mainly used for collection of blood and CSF.

v Pipettes:-

                 These are used for dispensing controlled quantities of



                             Various chemicals are used for preparation of solutions and reagents in a pathological laboratory. A chemical is a substance produced by a chemical process by various manufactures. It is of known compositions examples:- sodium carbonate, potassium hydroxide, hydrochloric acids, glacial acetic acid, etc.


                   It is a combination of two substances a solute and a solvent. The dissolved substance is called solute and most of the times the substance present in a relatively greater proportion in the solution is called the solvent.


                      This term can be applied in particular to any chemical compound or mixture of compounds, usually in solution, employed in chemical analysis or for the detection of biological constituents.


                                                                   A stock solution is a concentrated solution from which different types of working solutions can be prepared by simple dilutions.


                          Acids are proton donors and bases are proton acceptors.
                   Acid                  Base + H+  
          ALKALI: - these compounds yield hydroxyl ions on dissociation.

                       e.g.  NaOH                   Na+  +  OH-      

The various types of solutions and reagent used in a pathological laboratory are-
·       Normal solutions
·       Molar solutions
·       Percent solutions
·       Buffer solutions
·       Buffered substances
·       Indicators
·       Primary standards and
·       Other complex reagents.


The various types of equipments and instruments used in clinical biochemistry are as follows-
·       Balances
·       Hot plate and Magnetic stirrer
·       Centrifuges
·       Hot air oven
·       Incubators
·       Water Bathes
·       Photometers and Spectrophotometer
·        pH Meters    
·       Distillation plants
·       Deionizers
·       Cell Counter
·       ELISA Strip Reader
·       Automatic dispensers and diluters
·       Autoanalysers
·       Electrophoretic instruments
·       Osmometers
·       Electrolyte analyser
·       Semi-Automatic dry & liquid biochemistry analyser
·       Autoclave and microwalles
·        Acid –Base Analysers.



                                          The technician should be very well trained to-
1.  Approach the patient pleasantly and confidently.
2.   Obtain blood sample properly, quickly and without undue discomfort to the patient.
3.  Maintain proper record of collection.
4.  Handle the specimen carefully.
5.  Analise the specimen accurately.
6.  Maintain proper record of reports.


·       Pleasant personality.
·       Ability to speak well.
·       Alertness.
·       Enthusiasm.
·       Sincerity.
·       Working accurately and speed.
·       Ability to follow instructions and make corrections.



1.  Preparation for blood collection.
a) Each request for a blood specimen must include a number to identify all paper work and specimen associated with each patient.  The information given on the blood request form should be recorded on the specimen labels. 

Essential items include the following
                                                            I.        Patient’s complete name and age.
                                                         II.        Identification no.
                                                       III.        Date and time if necessary.
                                                      IV.        Name of physician ordering the tests.
b)  The specimen containers should be labeled appropriately before the specimen collection.

2.  Ascertaining  whether the patient has fasted
Some tests require the patient to fast. Such care is needed to ensure accurate results.
3.  Reassuring the patient.
The technician must gain the patient’s confidence
And assure him that, although the venipuncture will be slightly painful, it will be of short duration.

4.  Positioning  the patient
a)  The patient should be made to sit comfortably in a chair and should position his arm on a slanting arm rest, extending the arm straight from the shoulder and it should not bent at the elbow.

b)  If the patient wants to lie down, let the patient to lie comfortably on the back. The patient should extend the arm straight from the shoulder. For support, a pillow may be placed under the arm.

Requirement for blood collection

1.  Collection tubes, bulbs etc.
2.  Sterilized syringes and needles.
3.  Sprit or 70% ethanol.
4.  Cotton balls or gauze pads.


1.  Checking for paper works and tubes.
The tubes and bulbs should be checked for appropriate kinds and for paper labeling.

2.  Selecting vein site.
For most venipuncture procedures on adults, veins located in the arm are used. The median cubital vein is the one used for the patient.

  3. Following techniques are useful when encountering a            patient with difficult veins.
                 i.        Look for a blood drawing site.
               ii.        Feel for s vein using the tip of the finger. Think of four things when feeling for a vein, bounce, direction of vein, size of needle, and depth.
             iii.        Choose the vein that feels fullest.
             iv.        Try the other arm unless otherwise instructed.
              v.        Ask the patient to make a fist.
             vi.        Apply a tourniquet briefly.
           vii.        Massage the arm from the wrist to elbow.
         viii.        Cleansing the area to prevent any contamination. Sprit or 70% ethanol is used for cleansing and the area is allowed to dry to prevent possible heamolysis of the blood specimen.    

4. Performing the venipuncture
                 i.        The patient’s arm is gripped and thumb of another hand is used to draw skin taut.
                ii.        The vein is penetrated by positioning the needle at 30° to 40° angle.
              iii.        After the blood has been drawn the patient should release the fist and the tourniquet is also released.
             iv.        A cotton ball is held firmly over the venipuncture site as soon as the needle is removed.
               v.        Collected blood is dispensed in the appropriate tubes or bulbs.
             vi.        The blood in the anticoagulated bulbs is mixed carefully and blood collected in the tubes or bulbs without anticoagulants is kept on the room temperature.

5.  Separation of serum
                    I.        Allow the blood to clot.
                  II.        Loosen the clot slowly and centrifuge the supernatant fluid.
               III.        By using a Pasteur the pipette, separate the serum from blood cells and store it in a clean and dry test tube.





                       Billirubin is coupled with diazotized sulfanilic acid in the presence of caffeine to give an azo dye. No caffeine is added when “direct billirubin” is determined.


Total billirubin   :-    0.001 mg/dl
Direct billirubin :-     0.00-0.25 mg/dl

Sample material :-

                                  Serum, haparinised plasma or EDTA plasma.


Reagent 1.
Sulfanilic acid solution
Reagent 2.
Nitrite solution
Reagent 3.
Caffeine solution
Reagent 4.
Tartrate solution
Reagent 5.
Sodium chloride solution


      wavelenghth for total billirubin : 578 nm
        wavelenghth for direct billirubin : 546 nm
         cuvette                               : 1 cm path length
                  temperature                           : 20° C to 30° C
          measure against sample blank.

Total billirubin    
 Pipette into cuvette
Sample blank
Reagent 1.
Reagent 2.
Reagent 3.

Mix, let stand at 20°C to 30°C for 5min

Reagent 4
Mix, let stand at 20°C to 30°C for 5min

Read absorbance of sample against sample blank

 Direct billirubin:

 Pipette into cuvette
Sample blank
Reagent 1.
Reagent 2.
Reagent 5.

Mix, let stand at 20°C to 30°C for 5min

Read absorbance of sample against sample blank.



                      When a serum sample +ve for RF is mixed with latex reagent visible agglutination occurs. In sample negative for rheumatoid factor the latex remains in a smooth suspension form.


                                                                   i.            Allow  all reagents and samples to equiliberate to room temperature before use ensure that the test slide is clean and dry.
                                                                ii.            Place 50 µl of test serum/ controls on the circle of the test slide.
                                                             iii.            Add 2 drops of the latex reagent holding  the dropper vertically .
                                                              iv.            Use the mixing stick to mix and spread the reagents over vertically.
                                                                 v.            Gently rotate the slide for 2 minutes, and examine for agglutination immediately under bright light.

               RF LATEX REGENT:-

                                                Suspension of polystyrene latex particles of uniform size, coated with human gammaglobulines.



                                In 1901 ABO system was discovered by LANDSTEINER . the ABO system consist of four main groups-
1.   “A”
2.   “B”
3.   “AB”
4.   “O”
i.e. cell carrying A antigen, B antigen, both A and B antigen and cells that do not carry either A or B antigen respectively.
The blood group antigen react with their corresponding antibodies present in the serum of different blood group.


 The following conditions required for determination of ABO blood groups-

In organ transplantation.

In haemolytic diseases of the new born baby.

In forensic medicine.

* In malignant diseases.

In clinical medicine.


# Take a slide and then mark as A,B,O place an drop of antigen A in the area marked as A & one drop of anti B on the area marked as B & third drop of anti D on the area marked as O.

Disinfect the first two drops of blood.

# Add three drops of blood,  the area marked as A,B and O.

Mix the antiserum and cell with the stick or toothpicks.

# Observe the haemagglutination, record the result.



         HIV antigen are immobilized on a porous immunofiltration membrane. Sample and reagent passes through membrane and are absorbed into the underlying absorbent.
As the patient sample passes through the membrane, HIV antibodies, if present, bind to the immobilized antigen.
Conjugate bind to the Fc portion of the HIV antibodies to give distinct pinkish purple DOT against a white background.

HIV kit contains:

·       HIV TRI-DOT test device
·       Buffer solution
·       Protein A-Conjugate
·       -ve & +ve control
·       Sample dropper

Test procedure:-

1.  Add three drop of buffer solution to the center of the device (HIV TRI-DOT)
2.  Hold the dropper vertically and add one drop of patient serum using the sample dropper.
3.  Add five drops of buffer solution.
4. Add two drops of liquid conjugate directly from the conjugate vial.
5. Add five drops of buffer solution and read result.


1.  Non-reactive:- if only one DOT appears (control DOT) specimen is non-reactive for antibodies either HIV1 or HIV2.
2.  HIV2:-if two DOTs, one for control and other for HIV2 appear.
3.  HIV1:- if two DOTs one for control and other for HIV1 appear.
4.  If all three DOTs, one each for control, HIV1, HIV2 appears, the specimen is reactive for antibodies of HIV1 and HIV2.



Clinical significance:- 

                           Rapid-Widal is intended for the detection of Typhoid Ab usually found after two weeks of salmonella infection.
The level of the Ab progressively rise to a maximum by 3-4 weeks if no specific antibiotics are taken. A progressive rise in Ab titer during the period of infection is indicative of salmonella infection.


                Rapid widal is based on a reaction between specific species of killed salmonella core(O) Antigens, flagellar(H) Ag, paratyphi”AH”, paratyphi”BH” antigens and the Ab found in patient serum. By staining the bacteria in the Antigens enhance the visibility of the reaction on slide as well as test tube.


+ve control

Note:- All reagents contain killed bacterial Ag, preservatives and stabilizers


Clean the glass slide with distilled water and dry. Brings the Antigens to room temperature and mix well before use.

1.  Add sample a drop each to “o”,”H”,”AH” and”BH” circles.
2.  Add “O” Ag a drop to “O” circle, “H” Ag a drop to “H” circle, “AH” Ag a drop to “AH” circle, “BH” Ag a drop to “BH” circle.mix well using separate disposable plastic sticks.
3.  Observe agglutination within 5 min. under a bright light source.
4.  Run a –ve control using normal saline as sample.
5.  Run a +ve control using the +ve control as sample.


·       Observe agglutination in comparison with –ve & +ve control.
·       A progressive rise in titer is indicative of salmonella infection


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