Nuffield Research Summer Placement 2014
Norbrook Laboratories Ltd
Thermometric Validation Department
Project Title: Verification of Population, Identity and Purity of
Organisms for Biological Indicators.
Name: Aine Fearon
School: St Marys’ High School Newry
Year: 13
Age: 17 Years Old
Brief overview of my project
My project consisted of me completing spore strip verification of Bacillus atrophus.
Spore strip verification is carried out to compare the manufacture’s claimed
population against the actual population that is found in the results after testing has
been carried out. It is a bacteria that is used as a challenge organism in the sterilisation
of Oil base products, it is a GMP as well as an MHRA and FDA requirement that the
challenge be no less than 1.0 x 106 which must be independently verified by us in
order to say that the spore strips contain the claimed population.
Validations
Validation Departments are a legal requirement within this field of work, it must be
provided for Good Manufacturing Practice (GMP). The department is separated into
three parts, Thermometric, Chemistry and Qualification. This department of
Norbrook Laboratories Ltd does not necessarily make money for the business, but is
an extremely important part of the business as a whole. By not going through
validations, there is no actual proof, nor evidence that the equipment being used
entering the sterile suites are 100% sterile.
This means that they could be contaminating the products that will be used in treating
animals and humans. The compounds of the medicine could be changed as they are
adding unknown properties into the mixture. This could be extremely dangerous as
the amino acid sequence could be changed leading to new locations of hydrogen
bonds and di-sulphate bridges, this will change the shape of the active site and no
longer be complementary to the substrate as it was before. If there is a change in the
sequence it could lead to a mutation or a non-functioning protein, this can appear
through three different methods, substitution of a base, deletion of a base or an
addition of a base.
Tests are carried through many different methods to ensure that all equipment are up
to standard and fulfil the requirements of the protocol and the laws of the countries for
which the product will be entering. These tests include spiking of vials, spore strips
verification, calibrating pipettes, thermocouple wires and incubators etc. When
leaving the sterile suites the clothing which you are wearing is also checked through
the use of agar plates in different places to prove that you are still sterile by the time
that you are leaving the suite.
Introduction to project
The project provider is Norbrook Laboratories Ltd, which employees 40,000 people
worldwide. It is the only company outside the USA that can export and import to and
from the USA. It now produces human products as well as animal products. The
company has three factories within Northern Ireland as well as it very own farm and
one factory in the Republic of Ireland.
To work within the Norbrook Laboratories Ltd you must complete induction training
including health and safety and GMP, which contains paperwork at the end of each
stage. When placed in the workplace you must read all Standard Operating
Procedures (SOP), and then be trained by a senior in the actives that you will perform.
If not properly trained or if the senior does not feel as though you are adequate, you
will be unable to perform some of your duties, e.g.; take part in all trials.
When starting my project I had to firstly understand the aims and objectives. Why
complete this project? What is the context of the project? What is the next step of the
project? Where did the experiment come from? Where is it going in the future? and
What impact do the results have? I had to be able to verify that the independently
verified population of bacterial challenge is greater than or equal to 1.0 x 106 spores
per strip. That the verified population falls within the range of 50 – 300% of the
manufactures claimed population and also that the purity of the biological challenge is
at least 99% NOTE the manufacturers claimed population.
Spore strip verification is carried out to compare the manufacture’s claimed
population with the population that we receive. The manufacture claims a population
along with 100% purity of the challenge organism Bacillus atrophus. Bacillus
atrophus is used as a challenge organism in the sterilisation of Oil base products. It is
a GMP, MHRA and FDA requirement that the challenge be no less than 1.0 x 106 this
must be verified independently by us in order to be able to say with certainty that the
sterilisation process is sufficient to destroy the challenge organism. In short a
population of less than 1.0 x 106 spores per strip is an insufficient biological challenge
to the sterilisation process.
The use of this experiment in every day working life in the Thermometric Validation
Department is to independently verify the bacterial population of Bacillus atrophus
spore strips prior to their use as a biological challenge to the sterilisation of oil bases
used in the manufacture of sterile injectable products.
If the population is within the stated range as required by the regulatory authorities the
spore strips can be used in oil base sterilisation processes. This involves a challenge
of Bacillus atrophus being present to an oil base which has the exact composition as
one used in the production of our sterile injectables. The base will then be heated to
160ºC for a period in excess of 1 hour. 12 spore strips are used in each test.
Following the sterilisation the spore strips will be incubated in individual sterile
containers containing approximately 10-15ml of Tryptone soya broth (TSB) which is
a nutrient media designed to promote the growth of the challenge organism should it
survive the sterilisation process. They are incubated at a temperature of 30-35ºC for a
period of 7 days along with 2 positive controls (unsterilised spore strips) and 2
negative controls ( just TSB) they will be checked for growth each day for 7 days. At
the end of the incubation period the process is considered successful if the test spore
strips show no growth as do the negative controls. and the positive controls show
growth that is identified by gram stain and colony morphology as a gram positive
sporing rods, Bacillus atrophus, this will present as a purple rods under oil immersion
microscopy with the presence of a sub terminal spore- a small ball ¾ of the way down
the rods.
This project came from the fact that Bacillus atrophus is a thermophile (heat loving
bacteria), which is particularly resistant to conditions of dry heat, as opposed to steam
sterilisation, dry heat is what is used in an oil base sterilisation. It is an industry
standard bacterial challenge which is accepted as a method by all pharmaceutical
regulatory bodies, MHRA, FDA etc, there is therefore no plans for future
developments, as the process of developing a new method and having it accredited
would be extensive, expensive and there would be no assurance that it would be
successful.
There are both positive and negative impacts for this project; if the verification is
successful the biological challenge can be used in the validation of the sterilisation of
oil bases for use in the manufacture of sterile injectable medication. We can say with
confidence that the challenge we are presenting to the sterilisation process is sufficient
and that if this challenge is destroyed that the process is sufficient to destroy any bio
burden that may naturally be present in the composition of the base and active
ingredients prior to sterilisation. This is important as the oil base will be used as the
‘inactive base’ of the drug produced. As the drug will be an injectable there is no
margin for error in the sterilisation process as any presence of bacteria in the finished
product could be fatal.
Project in action
When beginning the project I needed to gather all the equipment that I would need
throughout the period of the project.
Equipment;

Laminar flow cabinet

Rack

Microscope

Dropper

Glass Slide

Sterile LAS water

Grams Crystal Violet

Lugaols Iodine

Grams differentiator (Acetone)

Grams Saframine Solution

Microscope immersion oil

Agar plates

Tweezers

Water bath

Empty test tubes/ sterilins

Glass beads 2mm

Test tubes with 9ml and 10ml of water

Spore strips

Wire loop

Gloves

Autoclave

Micro pipette

Manual pipette

Tips for both pipettes

Permanent marker

Autoclave tape

Vortex

Ice bath

Tray

Spreaders

Bunsen

Incubator

Temperature probe
Step 1
-
Fill four sterilins with ⅓ of glass beads
-
Tape together and place in a marked bag saying ‘ Validation please autoclave
caution glass’
-
Autoclave for two hours
-
Fill test tubes with 9ml of sterile water using manual pipette and replacing tip
if becomes contaminated , you need four sets of water with four test tubes of
9ml of water
-
Label Agar plates using permanent marker with 10-4 and 10-5 with spore strip
product number, you need two batches one of 10-4 and one of 10-5 with eight
plates each as you will be duplicating if not four plates as you take two
samples from each 10-4 and 10-5 for four sets of sterilins
-
When glass beads come out of the autoclave place 10ml of water into each
along with the spore strips
-
To do so you must take them to the laminar flow cabinet and use tweezers,
you must not touch the spore strip with hands only tweezers. Heat the tweezers
using the Bunsen then tear the seal on the packet, lift spore strip using
tweezers place into the sterilin.
-
Shake by hand and vortex the sterilins of glass beads and water till there is no
big bits of spore strips left.
-
Place in the water bath that it 80-85ºC for 10minutes.
-
Then place in ice bath for 5minutes.
-
Once again in the laminar flow cabinet with the Bunsen flame on, take using
the micro pipette 1ml of fluid from the sterilin with the glass beads and place
into the first test tube for that batch of 9ml of water shake and replace tip, take
1ml of this and place into the next 9ml of water, shake and replace tip, take
1ml of water from this and place into the next 9ml of water this is now 10-4 in
this test tube. Take 1ml of liquid from this and place into next 9ml of water
this is now your 10-5 .
-
Spread 1ml of the 10-4 and 10-5 onto two individual agar plates that are
labelled and use spreaders to spread the fluid, do not lean hard or it will
damage the agar on the plate, do not turn over until plate is once again dry.
-
Incubate for 48 hours.
Step 2
Count plates- you must count the colonies that are on the plates
You must then complete the calculation.
A) Sum of all biological indicators organism counts from each plate.
B) Sum of all 10-4 viable counts between 30 and 300.
C) Sum of all 10-5 viable counts between 30 and 300 multiple by 10.
D) Number of plates yielding 30 to 300 viable counts 10-4 and 10-5 .
Then: A+B ÷ C ÷100
If the calculation indicates that;
–
In house population is greater than or equal to 1.0 x 106 .
–
In house population is within -50% to +300% of label claim population.
–
Log in house population not less than 0.3 log from the label claim population.
–
Log in house population does not exceed log label claim by 0.48.
–
Biological indicator in house population complies
Once all these are answered as YES then this stage has passed
Step 3
- Label a sterilin with the batch number that has TSB only inside.
- Take one of the plates and this is the one that you will be working with.
- Using the Laminar flow cabinet and the Bunsen heat the wire hoop until it is red hot.
- Take the loop which is red hot, put on to the agar where there is no growth then,
swab using the red loop a swab of growth and put it into the Sterilin with the TSB,
repeat this three times with the same plate into the same Sterilin.
- Place into the incubator 30-35°C for 48 hours.
Step 4
Restreaking onto plates is the next step
- Label three plates with the batch number and three different temperatures.
- These will be the temperature of the incubators that they will be put into: 10-15°C,
30-35°C, 55-60°C.
- Using the Laminar flow cabinet and the Bunsen, heat the wire loop until it is red hot.
- Dip the red hoot loop into the test tube and put drops onto the plate.
- Where the drops are placed this then will be the most concentrated part of the plate,
use the loop to spread the liquid.
- From the point that you put the drops onto the plate swab horizontal .Take one end
and start the horizontal movement from there in a straight line and turn on a 90° angle.
Begin the horizontal swab at one end of your previous horizontal swab, repeat the 90°.
angle and swab horizontal again, once finished this line from the end do a wiggly line
into the middle of the plate.
- Incubate at the three different temperatures and leave for 48 hours.
Step 5
Gram staining is the next step.
-Firstly you must use the laminar flow cabinet with Bunsen lit.
- Label your glass slide.
-Flame wire (nicrom) loop until it is white hot.
-Use loop to put 3 to 4 loops of sterile water (LAS).
-Flame wire loop again and swipe a colony of the plate Geobacillus steartherphilus
which is a thermophile.
- Mix and spread with the sterile water on glass slide using wire loop.
- Put the glass slide over the Bunsen until it the water has dried; be care not to hold it.
for too long or the glass may crack.
- Go to the sink and use rack. Flood the slide with Gram Crystal Violet (purple) for
1minute.
-Risen with tap water.
-Flood the slide with Lugols Iodine (orange) for 1 minute.
-Risen with tap water.
-Flood the slide with Grams Differentiator (Acetone), do not leave on rinse of
immediately, water should run of slightly purple in colour.
- Flood the slide with Grams Safranine Solution (red) for 1 minute.
- Take the slides to the Microscope, with the oil lens use pH3.
- Microscopy immersion oil put 1 drop onto the slide.
- Look for a sub terminal spore- a small ball ¾ of the way down the rod.
(Tree shape).
- If there is this shape your trial has been successful and has passed.
Conclusion and Evaluation
I completed three batches in separate trials of different spore strips, only one batch
passed, two had failed once they reached step 2. As these two failed I repeated only
one to make sure that I had two trials that passed and I studied the other trial to
understand where I went wrong to prevent me making the same mistake again.
Like most experiments it could fail at any time and at any stage, if this does happen
the trial must be repeated from the start to get a more reliable result. Not only do the
trials have to be completed from the start, but so must the paper work as some of the
paper work will be incomplete because the trial could not be completed. You must
state in the first page or on an information record sheet attached on the front of the
document that it is a failed trial.
As I was completing the trials on an educational level my results were for learning
purpose and not for company use. I chose only to repeat one of the failed trials and
use the other for a learning experience. This is not allowed when you would be using
the trials for the company; each trial must be repeated until it does not fail. Even if the
trial passes up to step 5 and the end result is that it has not passed it must be repeated
in its entirety.
Although one of my trials passed first time, the one that had failed and was repeated
passed then too.
I am glad that I did repeat the test as it allowed me to improve and identify what could
go wrong in this experiment and how to avoid the same problem in my repeated trial.
My trials failed as when I added the 1ml of 10-4 and 10-5 liquid to the agar plates I
thought that they were dried and I turned them over to place them into the incubator.
Although they looked dried to me they were not completely dry which meant that
when I turned the agar plate over the liquid fell onto the lid of the plate instead of
staying on the agar, which left no colonies to grow on the agar. On some of the plates
the colonies had joined together and had grown in groups which resulted in not being
able to identify single colonies. This was an direct result of me turning the plate which
allowed the fluid to move across the plate in turn disrupting the colonies that had
began to grow on the agar.
On the repeat trial and to prevent the same mistake from happening again I left the
plates on the side of the bench for 45mins before turning the plates. Although I
believed that 45 minutes would allow them to dry, I didn’t turn the plates over before
putting them into the incubator, I lifted the plates and placed them into the incubator
therefore if they had appeared to have dried but not completely dry it would not
disrupt the liquid.
With taking this extra precaution the trial passed and the colonies grew perfectly on
the agar plates. This result allowed me to continue with the trial step 2 and I had no
other problems for the rest of the trial.
Although I had to repeat step 1 for one batch, I continued to work with the other steps
for the first batch that had passed, this meant that I did not upset the trial and reduce
the possibility of it failing by allowing the plates to be incubated for too long.
In the future when completing this trial, I believe that I could avoid all errors and pass
all trials first time.
My trials did prove that the manufactures claimed population was correct, that all my
aims and objectives were successfully met. That the claimed population is correct,
along with 100% purity. That Bacillus atrophus can be continued as the challenge
organism in the sterilisation of Oil base products. That the population is no less than
1.0 x 106 spores per strip is an insufficient biological challenge to the sterilisation
process.
I strongly believe that I have learnt a lot about not only the sterilisation process but w
how to actual carry out, complete and be successful in this type of testing.. I have
learnt many skills that will help me in my future career, skills ranging from being able
to measure liquids accurately, labelling correctly to avoid mix up in solutions or in
contaminating products. Labelling correctly prevents perfectly good products from
being discarded because the properties are unknown. Following procedures and
understand the next steps in the protocols. This has all helped me as in the future I
may find myself re-using the skills that I have developed.
Reference
Standard operating procedures SWVA-010-13
Microbiology lab manual version 2.0
Applied and environmental Microbiology, Vol 36, No 5, pg 715-717 November 1978
Acknowledgements
I would like to give my sincere thanks to St Mary's High School Newry, especially to
the Year Head Mr Fitzpatrick for making me aware of the Nuffield Project. Thanks
also to Careers teacher Mrs Kidd who guided me through the application process
resulting in my application being successful.
Sincere thanks also to The Nuffield Research Placement programme that presented
me with the brilliant opportunity at an early stage in my life.
I would like to thank Norbrook Laboratories Ltd for allowing me to complete my
placement within the company.
I would also like to thank Sandra Crusnan for being my supervisor throughout my
placement.
Most importantly I would like to thank the Validation team:
-Sharon Watson,
-Ruth Clarke,
-Laurie Lee,
-Carla Caiceo-Wolleter,
-Lauran Gallagher,
-Aislinn Mc Cavigan,
-Richard Craven,
-Conor Breen,
-Zabrina Lilley,
-Brian Cotter,
-Vicky Magowan.
This group of people made my placement a very pleasant environment to be working
and learning in. They were extremely warm and welcoming. I would not have been
able to complete my project without their support and their guidance, and would not
have been able to reach my full potential in the short space of time I had with them,
without the team.
I fully enjoyed working with everyone and appreciate the time and energy they gave
to me and my project. I would love to come back again and work with the team once
more in my future. The team is full of hard working people that are fully dedicated to
their jobs and have a passion for all that they do. I hope that they enjoyed the time
that I spent in the department as much as I did.
I cannot express how much I have enjoyed my time with you.
Many Thanks.