Sterile formulation basics

Question 1

Sterile key quality attributes

Answer 1

Free from micro, endotoxins and foreign bodies
Evidence products was made to GMP
Correct packaging and labelling
Assurance of stability across shelf-life
Accurate potency, correct formulation & isotonicity
Container closure integrity
Sterile BYPASS body’s defences, onset rapid & Irreversible

Question 2

Types of sterilisation: (EMA guidance decision tree)

Answer 2

Moist heat (Steam): 15 mins at 121oC or 3mins at 134oC
Dry heat: 2 hours at 160oC or 1 hr at 170oC
Depyrogenation: 30mins at 250oC (3 log reduction in ET)
Depyrogenation tunnel: 300oC/5min or 320oC/3min
Aseptic (Filtration): 0.2um filter
Gamma Irradiation: 25kGy, Cobalt, low penetration, requires direct contact, distance from source critical
Ethylene Oxide: requires direct contact, only use if no alternative, must ensure all traces of EtO removed

Question 3

Biological Indicators (6 log reduction) / Chemical Indicators:

Answer 3

Steam: Geobacillus Steareothermophilus
Dry heat/Ethylene Oxide: Bacillus Atrophaeus (Bacillus Subtilis)
Depyrogenation: Endotoxin (3 log reduction)
Aseptic: Brevundomonas diminuta
Gamma Irradiation: Bacillus Pumilus
Chemical Sanitisation: Bacillus Atrophaeus
Optical Dosimeter: Gamma Irradiation  colour change
Bowie Dik Test: Steam Penetration  colour change
Autoclave/Indicator Tape: Steam (temp)/Gas (exposure)  colour change

Question 4

Sterility Assurance Level(SAL):

Answer 4

Prob. of there being one viable m/org in container. Min: 1 x 10-6

Question 5

Key sterilisation terms

Answer 5

D-value: Time taken to reduce population by 90% (1 log).
By knowing D and time of sterilisation cycle can predict the LRV and SAL
Z- Value: Temp change which results in a 90% change in the D value.
Allows comparison of killing effect of sterilising cycles
F-value: Time at steady state reference conditions having the same lethal effect. Establish different but equivalent cycles.
F0 Value: Steam Sterilisation equivalent time in minutes at 121oC.
1 minute at 121oC – 1 F0 Unit (EU req F0 of at least 8 (i.e. 8 mins at 121oC))

Question 6

Terminal Sterilisation (FILL  STERILISE):

Answer 6

Product sterilised in final container (typically moist heat)
Filled under Grade C c/room conditions using aseptic tech.
Entire batch sterilised in autoclave/oven (steam/dry heat)
Validated as 6 log reduction of bioburden (moist heat BI - Geobacillus stereothermophilus)
Not suitable for all product types, e.g. biologicals
Product: WEIGH  MIX  FILTER  FILL/CLOSE  STERILISE  INSPECT  LABEL/PACK
Container: WASH  STERILISE/DEPYROGENATE (HEAT TUNNEL/DEPYROGENATION CYCLE IN OVEN)

Question 7

Sterility

Answer 7

Absence of all viable m/orgs

Question 8

Sterilisation by Filtration and Aseptic Processing (STERILISE  FILL):

Answer 8

Assembled & filled using sterile ingredients & components
Filled under Grade A c/room conditions using aseptic tech.
Bulk soln made sterile by filtration through 0.2um filter (LRV > 10)
NB to ensure bulk soln bioburden as low as possible prior to filtration
EMA guidance 10CFU/100ml for pre-sterilisation filter bioburden
Filter integrity tested pre and post use (PUPSIT)
Filtration validated as 6 log reduction of bioburden (Brevundomonas diminuta)
Process validated using aseptic simulations & other control/monitoring techniq.
C/rooms and aseptic behaviours critical
Product: WEIGH  MIX  STERILE FILTER, FILL/CLOSE  INSPECT  LABEL/PACK
Components depyrogenated
NOTE: Components generally bought sterile so do not req sterilisation / depyrogenation steps.
FIT: Bubble Point Test
Filter wetted, impermeable to gas at low pressure. As pressure increased gas is forced through & stream of bubbles observed. If filter is damaged a lower pressure of gas is required to get to the same point.
Forward Flow Bubble Test:
Membrane wetted, pressure applied (60-80% of BP) and due to differential pressure gas will diffuse across membrane. Damaged filters allow gas to diffuse at pressures below the Bubble Point.

Question 9

Autoclave Validation/PQ

Answer 9

comprised of at least 3 repeats for each defined load. The critical aspect is to validate sterility in each of the loads.
Full PQ every year, and after significant changes – detail in annex 1
Objective: prove sterilisation of specified load. Define SAL, and where sourced from (Annex 1)
Procedure:
Type of cycle (porous/fluid) including all critical parameters (air removal, steam penetration)
Equipment needed for the test, inc. thermocouple to verify temperature
Temperature mapping – empty chamber. Identify ‘worst-case’ areas for loaded cycles.
Number and location of each item in the load (minimum and maximum)
Locations of the BIs and datalogger probes should be placed in “worst-case” locations in the load to provide assurance that the entire load is sterilised
A step-by-step listing of how to carry out the test, e.g. from SOP
Acceptance Criteria: lists the required result or range of results. Examples of acceptance criteria for PQ involve sterility and temperature (e.g. +/- 2 degrees from set point). For BIs, there should be no surviving spores.
Result Record (test script): All test data recorded. At least 3 iterations runs for process tests. Any failure to comply with the acceptance criteria makes a failed test. The record must include:
Autoclave tape / autoclave print out to ensure temp/pressure. Compare against standard chart.
Thermocouple result.
BI results.
Datalogger results and their compliance with the Acceptance Criteria.
Verification: results reviewed for compliance with acceptance criteria. Review is finalised with signatures from the person who ran the test & countersigned by second person.
A well-executed PQ enables you to demonstrate and document that the autoclave can execute the desired processes and provide the SAL required for your process.
PQ is generally the final step in qualification. This testing creates a baseline level of proof of the autoclaves ability to do the specified job, and is basis for requal. at periodic intervals.

Routine Operation:
Validated loading patterns inc. logbook for use of autoclave
Calibration and maintenance of critical devices  defined schedule, PQ after changes
Review of cycle traces
Annual revalidation/PQ  ongoing process verification

Question 10

Steam Sterilisation (EN285 and EN554):

Answer 10

Key aspects: Steam quality, time, temperature (2 temp probes – 1 monitor/1 control), pressure, air removal, equip & cycle design, validation, operation, maint & cal
Removes air and replaces with steam, pulses on trace during air removal
Kills by energy transfer
DOES NOT DEPYROGENATE  depyrogenation step required (inactivation/removal)
Condensate drained
Doors each side to help contamination control
Steam Quality for Autoclaves:
Physical aspects (EN285) include:
-Temperature (not superheated),
- Dryness (liquid water content),
-Non-condensable gas content.

Chemical aspects include:
- Chemical/Microbiological quality

Deviations from established ranges can result in issues:
Wet loads (bad as autoclave bags not porous if wet)
Damaged loads
Unsterile loads
Sterilisation (biological and chemical) indicator failure

Question 11

Dry Heat Sterilisation/Depyrog:

Answer 11

CPPs: Time & Temp
Fan to circulate hot air
Air HEPA filtered in & out

Question 12

Porous load = equipment

Answer 12

Items in autoclave bags, essential all air removed & steam allowed to penetrate all surfaces. Air removal/steam penetration difficult, part. for filters & tubing  vacuum air removal & vacuum/pressure steam pulsing req’d. PULSES CRITICAL

Question 13

Fluid loads = ampoules/vials

Answer 13

Simpler cycle, doesn’t rely on direct contact of steam, but by generating steam atmosphere in ampoule/vial via energy transfer from autoclave steam through container walls. Removal of air therefore not as critical. PULSES NOT REQUIRED.

Question 14

Sterilising/Depyrogenation Tunnels

Answer 14

3log reduction in endotoxin  also meet SAL  depyrogenation tunnel will sterilise & depyrogenate
Combo of vial washing & product filling machine used
Cooling zone: prevent product cooling too quickly
Laminar air flow  air flow NB
Particulate measurement NB
Maintain differential pressures
HEPA Filter DOP testing – on in/out filters

Question 15

Requirements for sterile manufacture - Designing Process:

Answer 15

Facility Design
Clean room classification & monitoring: ISO 14644, Annex 1. Cleanrooms must be QUALIFIED and VALIDATED.
Environmental Monitoring
Utilities & Services inc. HVAC system (Air/Pressure Cascades/Air Changes), Water
Equipment design
Room finishes: cleanable, non-shedding, durable, continuous
Bioburden Control
Material and people flow
Access Control
People
Training inc. monitoring of training effectiveness
Gowning
Number of people

Question 16

Clean rooms design

Answer 16

Purpose: Create controlled environment (temp, RH, m/orgs, particles) in diff zones & provide layers of protection (Grade D  C  B  A), prevent contam re-entering
Filtered Air: HEPA filters
Typical pressure diffs: 10 to 15 Pascals (magnahelic gauges/light alert system)
Positive Pressure: Air pressure in room greater than surrounding area
Protect product in the room. Air flows to lower pressure areas.
Negative Pressure: Air pressure in room is lower than surrounding area
Used to keep any possible contamination in room
Typical Air change rates: At least 20 per hour, recovery time 15-20mins
Depends on what you have qual & validated – Smoke studies
In Grade A can be 100 per hour
C/room design considerations
Facilities for People Prep
Equipment & Component Prep Facilities
Product Prep Facilities
Interfacing equipment to c/rooms
Transfer in/out of c/rooms
Relationships between areas

Question 17

Cleanroom control

Answer 17

Filtered Air
Differential Pressures
Air Changes
Remove potential sources of contam.
Training
Gowning
Controlled access
Zoning (A/B/C/D)
Controlled environ

Question 18

Controlled areas

Answer 18

Grade A/B
No drains
No WFI: must be sterilised into area
Materials entering sterile  double door autoclaves / unwrapped and transferred via transfer hatch

Backgrounds:
Grade A Isolator: At least Grade D
Grade A RABS (Restricted Access Barriers)/other (LAF): Grade B
BFS (aseptic): QRM, at least Grade C with Grade A/B gowning

Question 19

RABs

Answer 19

Open/Closed system
Dedicated air system/exhaust to room, HEPA filtered air
Laminar air flow
Gloveports
Open/Closed operation  door open, Grade A
No integrated decontam. system  manual clean.
Not for highly potent/toxic products

Question 20

Changing Rooms

Answer 20

Airlocks
Annex 1 give gowning req’s
Must have enough space
Flushed with filtered air
Grade A/B multi stage change
Final stage of change room should be same grade (in at-rest state) as the grade it leads into.
Separate entry/exit rooms
No sinks in Grade A/B
Monitor operators on way out

Question 21

Isolators:

Answer 21

Sealed system with controlled environment
Dedicated air supply and exhaust with HEPA filtered air
Transfer hatches
Air flow uni-directional  smoke tests
Gloves: Pre & post use integrity testing, leak testing
HEPA filters: Integrity tested
Air quality in isolator measured  particulates
Automated decontam. of interior using VHP after close-up

Question 22

Clean Room QUALIFICATION / VALIDATION - ISO 14644

Answer 22

Specifies classes of air cleanliness as number of particles expressed as a concentration in air volume.
Also the standard method of testing to determine cleanliness class, including selection of sampling locations.

DQ / IQ / Demo of classification / OQ / PQ/ PV
Design factors:
Product type – Aseptic/terminal
Capacity, storage needs
Eqp, Services & Maintenance– power, gases, water
Regs – GMP & safety
People/material flows (waste removal)

IQ:
Verify facility has been built to DS

OQ: As Built (all services, but no equipment) & At Rest
Verify correct functioning of
cleanroom – especially HVAC
Smoke studies (video!)
Filter integrity testing
Recovery tests
Airflow (air changes/velocity)
Physical & Micro air/surfaces quality (particle / micro counts)
PQ: In Operation (simulated production conditions)
Verify cleanroom conditions under simulated production controls
Physical & Micro air quality (particle / micro counts)
Airflow (Air changes/velocity/smoke tests)
HVAC - Temp/humidity/pressure diffs
PV / On-going operation/maintenance:
Regular environmental monitoring of at rest and operation air quality
Media fills
Cleaning/Disinfectant – rotation of sporicidal /disinfectant
HEPA filters – DOP every 6 months
Airflow – air changes/velocity/smoke tests
Staff behaviours

Question 23

Environmental Monitoring – PDA Guidance

Answer 23

Non-viable particles:
Continuous counting in Grade A, recommend continuous for Grade B
Number of sampling sites taken from ISO14644

Viable Particles (Micro):
Active Air (air sampler)
Passive (Settle plates)
Usually TSA
Exposed to atmosphere will collect particles & micro
Typically exposed for up to 4 hours  drying risk
Contact plates
Surfaces
Finger dabs

Exit monitoring:
Contact plates taken from locations on operators gowning.

TRENDING CRITCAL in environmental monitoring.
Set Alert & Action limits

Set up a Environmental Monitoring programme based on:
QRM.
Look at smoke studies. Worst / high risk areas.
Facility / Eqp / Processes
Process inputs
Risk assessment periodically reviewed for effectiveness

Question 24

DOP (Dispersed oil particulates): Used to test HVAC filter integrity.

Answer 24

Oil dispersed as aerosol into upstream flow of filter. Number of particles in downstream flow measured using a calibrated photometer.
Level of challenge typically 50-100ugs/l, leak defined as >0.01% of upstream challenge.
Trend differential pressures to show any issues with filters (holes/blockages).

Question 25

Process Simulations (Media Fills) – Validation of aseptic processing

Answer 25

Annex 1 and PIC/S (more detailed) guidance
Should imitate as closely as possible the actual process, including set-up, and include all critical subsequent steps in your aseptic filling operation.
Choose a suitable media (to represent your product).
Take into account interventions known to occur during normal production, as well as worse-case situations.
Must cover all shifts and operators. Operators only considered trained and qualified if they have taken part in a successful media fill.
Must be performed as initial validation with 3 consecutive satisfactory simulations and after any significant modifications (e.g HVAC, equipment, water changes).
Number of vials to be filled depends on validated batch size (Annex 1)
Batch size <1,000 – perform media fill on full batch size.
Batch size 3,000-100,000 – perform media fill on 3,000-10,000 units
Batch size >100,000 units – perform media fill on 10,000 units
New Annex 1: Batch sizes under 5,000 – no. containers for media fill should at least equal prodn batch size. For larger batches typically a min of 5,000 – 10,000 containers are filled.
Vials should be filled with enough media that when rotated the media touches all of the inner surfaces of the vial.
Risk assessment may be used to determine the worse case vial size for products with multiple vial sizes. Bigger, wide neck vials generally considered high risk, as easier for ‘things’ to get in the vial.
Video and or have scribes during media simulations. This is helpful to look back at if failures occur.
If failures occur, investigate, look back at all batches released since the last successful media fill.
Performed at least every 6 months (per line & per shift), but based on the risk of your product/process (e.g. radiopharmaceuticals may be done more often due to RTRT used and reliance on QMS and processes).

Question 26

How would you plan a media fill?

Answer 26

Perform RA & review process, identify worst case parameters (e.g. longest fill duration, widest container opening, longest exposure time of open container). Document and execute under approved protocol. Protocol to include all aspects of media fill, including acceptance criteria.

Interventions:
Defined & classified regarding contamination risk, frequency etc.
Covered during media fill
Duration of media fill planned to allow performing all defined interventions
Proper documentation of interventions required (batch records)

Other points to consider:
Shift patterns
Fill level of units
Max. holding time
All integral units to be incubated

Incubation / storage
Different possibilities e.g.:
14 days at 20-25°C / 7 days at 20-25°C and 7 days at 30-35°C (Heat up period to be considered). Inspect after each incubation period.

Evaluation/Examination
To be performed or at least supervised by QC personnel
Examination by educated, trained and experienced personnel
Growth promotion to be demonstrated for medium

Question 27

Media fill pass criteria

Answer 27

Media fill pass criteria
The objective of the media fill is to produce zero contaminated units, irrespective of run size. Therefore the target involving such simulations should be zero positive units.
New Annex 1: No set criteria – target zero growth. Any contaminated unit should result in failed process simulation and investigation performed. Sufficient no. of successful, consecutive repeat media fills performed to demonstrate process returned to state of control (normally min. 3)

Discovery of any positive units, investigate with RA to assess any potential root causes, CAPAs and respective documentation.
ID micro
Find what batches have been manufactured since last successful media fill. Quarantine what’s in your control.

Following any failures, it is critical to verify the robustness of CAPA, may need to perform multiple media fill runs to do so
* Investigations which determine a definitive and readily identifiable root cause, might provide grounds for a reduced number of repeat run(s)  CAPAs should be put in place to avoid such issues and deviations to studies and processes from reoccurring.
* Execution of media fills without investigation to identify and correct any potential root causes is not acceptable.

Question 28

Blow-Fill-Seal (BFS) – Annex 1

Answer 28

Determination of “Critical Zone” (Grade A)
Environmental background, based on QRM.
Terminally sterilised at least Grade D
Aseptic, grade C with gowning as A/B
Validation & reproducibility of CIP and SIP KEY!
Eqp design & Qual
Integrity testing of product pathways
Duration of batch or filling campaigns
Resin control
Care with interventions into the critical zone

BFS
Forming, filling and sealing of container in a continuous process
Liquid filled containers

Question 29

Lyophilisation: ‘freeze drying’ of a product, usually to enhance stability

Answer 29

Sublimation of ice to vapour at low pressure
Tg – Glass transition temp – Critical temp for primary & secondary drying phases
Shelf loading (High Risk as vials not completely sealed – Lyo caps)
Load top down to avoid contamination into vials
Validated loading pattern
Shelf Freezing
5oC down to -60oC over 1-2 hours at 1 bar
Primary Drying
Sublimation of ice to vapour, 5oC
Below Tg for 12- 72h 1-5mbar
Secondary Drying
De-sorption of bound water, >20oC
Below Tg for 6 – 24h, 0.01mBar
Container Sealing
Typically 25oC, N2 1 bar or below
Shelf moves up to push caps fully into vials
Vial overseals completed after vials taken from chamber  vials kept under Grade A.
Chamber must be SIP before use - 15 mins at 121oC. CIP after use  inside product contact
Defrosting of condenser – must be cleaned regularly to avoid cross contamination
N2 and air enter through Hydrophobic filters.
Speed of freezing determines size of ice crystals  critical to structure of ‘cake’ and stability of product  slow freezing best, gives larger ice crystals
Equipment exposed to extreme temperatures & pressures  leaks tests NB

Question 30

Freeze Drying

Answer 30

Prepare stable products from materials unstable in soln / sensitive to heat, e.g. antibiotics, proteins