IC8-9 Sterilization Process

Question 1

What is SAL? What does it indicate?

Answer 1

Sterility Assurance Level.

The probability of one viable microorganism in a certain number of drug products (1 in 10^6 is an acceptable safety level according to pharmacopeial standards)

Question 2

What are 3 criteria of sterility?

Answer 2

No microorganisms

Endotoxins within limit

No detectable particles in vial (Not cloudy)

Question 3

Sterility is a general requirement for?

Answer 3

• Parenteral preparations
• Eye preparations (ophthalmics)
• Preparations for irrigation

Question 4

Why are sterilization processes not a replacement for process development?

Answer 4

Before sterilization, bioburden must be minimized even in manufacturing

Question 5

What is bioburden?

Why is high bioburden critical?

Answer 5

The initial microbial load

• Higher risk of contamination with viable microorganisms.
• Higher risk for contamination with pyrogens.

Question 6

What are the most sensitive steps for sterility?

Answer 6

1. Equipment must always be working
2. Practices and Training
3. Weather Environment can affect filtration systems such as the exhaust
4. Personnel
5. Engineering

Question 7

What is inactivation factor?

Answer 7

The degree to which the viable organisms is reduced by the sterilization treatment applied

Question 8

For overkill approach, a sterilization process is applied such that it reduces the Biological
Indicator (a particular microorganism) by _____. If you do this method, what are the advantages and disadvantages?

Answer 8

a factor of 10^12 (12 Log Reduction in Microbial Load)

Advantage - Safe product
Disadvantage - Material may not withstand harsh sterilization process

Question 9

An SAL of 6 log reduction is sometimes referred to as ________________. 4 log reduction is _________ and 3 log reduction is _________

Answer 9

Parenteral Sterilization; High sterilization; Low sterilization

Question 10

What tests can be done on the product to ensure SAL is met?

Which phases of the manufacturing procedure are these tests done?

Answer 10

Final Product Test
- Validation of Sterility Test (Suitability Test, Growth Promotion Test)
- Endotoxin Test
- Sub-visible particle Test (Light Obscuration Particle Count or Microscopic Particle Count)

Process Testing
- Bioburden IPC Test

Question 11

How is the suitability test performed?
What result should you expect after incubating the tests for 3-5 days?

Answer 11

• Spike with known quantity (<100 cfu) of known microorganisms (Positive Control)
• Normal product (Negative Control)

If the product is NOT sterile, the turbidity of the test sample in the negative control = the turbidity of the positive control

Question 12

What is an important consideration for suitability test?

Answer 12

How many test samples we are going to use

Question 13

What is the main source of contamination? How do we reduce this source of contamination?

Answer 13

People

Isolator (Closed fume hood)
1. Protect operator - Negative pressure inside
2. Protect product - Air goes out (Higher pressure inside than outside)

Question 14

What is the purpose of growth promotion test? What is the procedure for growth promotion test?

Answer 14

To confirm that each lot of growth media in the sterility test procedure will support less than 100 viable microbial growth

Culture media
1. If cannot support growth (<100), test fail (False negative - Microbes cannot grow because of medium)
2. If media is non-sterile, test fail (False positive - Microbes grow but not due to product but externals)

Question 15

What is the purpose of suitability and growth promotion test in the validation of sterility?

Answer 15

1. Suitability test - Ensures final product is truly sterile
2. Growth promotion test - Ensures that the growth medium used to determine the sterility of final product is reliable (fit for purpose) to produce consistent results - Makes sure that there are no false results

Question 16

What dictates the choice of microorganism to be tested in the final product?

Answer 16

Final Product

Environmental contamination (Medium comes after you select the strain)

Question 17

What is the purpose of endotoxin test? What is the most common endotoxin test? How does it work

Answer 17

To make sure that injectables are endotoxin free (LPS found in Gram negs)

Limulus Amoebocyte Lysate (LAL) Test - Clottable protein & LPS react (From horseshoe crab blood lysis)

Question 18

What is the endotoxin limit concentration (equation)?

Answer 18

ELC = K/M (Ratio of Maximum endotoxin dose to Maximum recommended dose)

Question 19

What is the maximum valid dilution (MVD) and why is it important?

Answer 19

MVD = ELC / Method sensitivity

If you overdilute above the MVD, you may get false negative because you think there is no endotoxin but it is really due to dilution

Question 20

What is bioburden in-process (IPC) Testing?

Answer 20

The concentration of microorganism in a material (Total number of organisms per ml or per gram)

Question 21

Why is Bioburden testing important?

Answer 21

• Need to control the microbial load at every step of manufacturing
• Need to control microbial load at the sterile filtration step in the manufacturing of sterile biologic drug products before aseptic processing

Note Process: Sterile filtration -> Aseptic filling & Processing

Question 22

What is the accepted limit for number of CFU and sample test volume for EMA guidelines for Bioburden Test?

Answer 22

Not more than 10 CFU / 100 ml

Question 23

What are some ways to reduce bioburden?

Answer 23

Filters:
- Pre-filters: Reduce microbial load prior to and on sterile filter
- Larger Single filter / Multiple filters in series: Effective filter surface area
- Sterile filter membranes with high microbial retention capabilities (≥106 CFU/cm2)

Others:
- Limit hold times and room temperature storage
- Aseptic handling techniques
- Limit batch volume to be sterile filtered
- Test integrity of sterilising filters pre- and post-use

Question 24

How to calculate bioburden?

Answer 24

Find the number of cells surviving at time t given the initial number of cells (N0) are declining at a constant death rate (k) for a period of t time

LogN = Log N0 – k*t

Question 25

What is D-value and its relation to bioburden?

How do we calculate the time to achieve sterility assurance?

Answer 25

Decimal reduction value = Time exposure required from a sterilization process to reduce a microbial population by 90%
LogN = Log N0 – kt
Log N/N0 = - kt
Log 1/10 = -k*D
D = 1/K
t = D * (Log N0 – log SAL)

N can be substituted with the SAL required

Question 26

Light Obscuration Particle Count Test: The apparatus is calibrated using dispersions of spherical particles of known sizes between ______________

Answer 26

10 and 25 μm

Question 27

Light Obscuration Particle Test:
For small-volume parenterals less than 25 ml in volume, the contents of __ or more units are combined in a cleaned container to obtain a volume of not less than ____

Answer 27

10; 25 ml

Question 28

Light Obscuration Particle Count Test:
The preparation complies with the test if the average number of particles present in the units tested does not exceed ____ per container equal to or greater than _____ and does not exceed ___ per container equal to or greater than ____.

Answer 28

6000; 10 μm

600; 25 μm

Question 29

2 Sterile Manufacturing Methods

Answer 29

1. Aseptic Filtration
2. Lyophilization

Question 30

What is aseptic process simulation?

Answer 30

A method of validating an aseptic process using a microbiological growth medium as substitute for product and employing method, that closely approximate those used during the drug product process.

Question 31

What is the purpose of aseptic process simulation?

Answer 31

To qualify the process at the beginning and to confirm its aseptic capability periodically.
- Assess machine assembly, standard interventions, handling & storage of components etc.

Question 32

What is one drawback about aseptic process simulation in qualifying the process?

Answer 32

May not be enough to evaluate the whole process because at any “point-in-time” there may be an issue but you cannot be sure which point is failing

Question 33

Is aseptic process manufacturing a QA method? So what is required in assessment?

Answer 33

No. Therefore, a full validation of all sub-processes is needed. Need risk assessment as well

Question 34

What is the filter size for aseptic (sterile) filtration?

What is this method applied to and what can it remove?

For the things it cannot remove, what may be needed?

What criteria of removal does aseptic filtration follow?

Answer 34

0.22 μm;

Thermolable objects;

Pyrogens (Bacteria, Moulds but not all viruses or mycoplasmas);

Additional heat treatment

A standard test organism with minimum concentration of 10^7 cfu/cm^2

Question 35

What should be in you growth medium compounding solution preparation in the aseptic process simulation?

Answer 35

• Buffer / Growth medium replaces final product
• Sterilization of medium
• Place medium in contact with every surface of equipment IN THE PROCESS (must go through the system even longer)
• Filter functionality test

Question 36

What is lyophilisation?

Answer 36

Freeze drying - Frozen product placed under vacuum and changed to vapor to remove water

Question 37

Lyophilization process 7 steps

Answer 37

1. Dissolve in water for injection
2. Sterile filtration 0.22 micron
3. Filling into sterile containers + Partial stoppering in aseptic conditions
4. Lyophilizer & Load into chamber
5. Freeze on cooled shelves in freeze-drying chamber
6. Vacuum and Heat
7. Complete stopper (Hydraulic / Screw Rod)

Question 38

3 Separate interdependent processes in lyophilization & what issues can be encountered at each step?

Answer 38

1. Freeze - Crystal size polymorphism
2. Primary drying (sublimation) - Temperature rise indicates endpoint
3. Secondary drying (desorption) - Water content in crystals < 1% as endpoint

Question 39

Other alternatives to lyophilization

Answer 39

1. Sterile Recrystallization - Crystallize in solution then filter
2. Spray drying - Solution sprayed in vacuum to get fine powder (Inhaler products)