Stopper Movement, Gas Bubbles in Shipping and Improving the Container Closure Integrity of a Pre-filled Syringe
Shawn Kinney, PhDPresident, Hyaluron Contract Manufacturing
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Stoppers: Syringe stopper is not held in place
• It has been shown that the stopper in a syringe containing a gas bubble will move in response to changes in ambient pressure
• Amount of movement is proportional to:– Size of the gas bubble– Pressure differential between pressure at which the syringe was
filled and the external pressure
• Most pre-filled syringes will be exposed to reduced pressure multiple times during their lifetime. – Airline shipment exposes syringes to reduced pressure
equivalent to elevation of approx. 8,000 ft. (approx. 2,500 m)– A gas bubble increases in volume by 36% at this elevation
Parameter: HsbHCM defines a parameter, Hsb, (the height of the sterile barrier) which is the distance from the lower most to upper most point of intimate contact the stopper makes with the syringe
Multiple stopper movement
• Expansion of the gas bubble may cause a sterility failure if the stopper moves a distance greater than Hsb
• Same effect (sterility failure) could occur if a stopper moves multiple times less than Hsb, if the sum of all of the stopper movements exceed Hsb
Is it possible for microorganisms to be mechanically carried from the non-sterile side of the stopper to the sterile product?
Bacterial CCI challenge under simulated shipping conditions
• Syringe– 3 ml pre-filled syringe filled with sterile 3 ml TSB– standard stopper– 3 mm air bubble
• Challenge - bacterial spores above the stopper • Control - no bacterial spores – 100 units each• Pressure history
– Several days room temp ambient pressure– 2 days with 5 exposures to reduced pressure
(17,000 ft, ~5,200m elevation), room temperature
Syringe: 3 mm gas space
Bacterial Challenge Selection
• Spore– Smaller than vegetative forms– Desire hydrophobic spore– Spore is more likely of concern than vegetative form– Available in a liquid– Requiring higher temperatures to prevent growth until
incubation
• Geobacillus stearothermophilus– Available in vials from Raven Biologics at 106 spores/ml– 1 – 1.5 µ diameter vegetative, spore smaller– Approx. 60 °C optimal temperature
Placing challenge solution in the syringe
• Approx. 10 µl spore solution placed above first stopper rib
• 4 x 106 spores/ml X 0.01 ml
• Approx. 40,000 spores per syringe !!
Hsb = 6 mm
Test Equipment
Test Equipment
Exposure history of syringe after challenge• 10 days room temperature, ambient pressure• Simulated flight with 5 exposures to reduced pressure• 17” Hg (569 mbar ) vacuum equates to pressure at 17,000 ft
(5,200 m) elevation• Gas bubble expands from 3 mm to approx 5.8 mm in height
(7 mm theoretical)• Total movement 14 mm, 233% of Hsb!!!
Exposure # Duration exposure to 17” Hg vacuum
Recovery at ambient
1 2 hr 16 min 1 hr 35 min
2 1 hr 48 min 47 min
3 1 hr 47 min 1 hr 10 min
4 2 hr 23 min 1 hr 36 min
5 40 min To incubation
Results
• Incubated units at 55ºC for 48 hours
• Challenge units – 14/100 positive
• Control units– 0/100 positive
Results
• Incubated units at 55ºC for 48 hours
• Challenge units – 14/100 positive
• Control units– 0/100 positive
Results
• All positive units, presumptive ID
• GeobacillusStearothermophilus
Results
• Placed approx. 0.4 ml of TSB above stopper of negative challenge units
• All showed growth after 2 days at 55°C
• No growth below the stopper was observed
• All positive units, presumptive ID: GeobacillusStearothermophilusabove stopper
Repeated experiment
• Syringe– 3 ml pre-filled syringe filled with sterile 3 ml TSB– standard stopper– 3 mm air bubble
• Challenge - bacterial spores above the top of the stopper
• Control – bacterial spores above the top of the stopper
• Dried overnight
Placing challenge solution in the syringe
• Approx. 20 µl spore solution placed on top of the stopper
• 6 – 8 small drops, total of approx. 20 mg spore solution
• 4 x 106 spores/ml X 0.02 ml• Approx. 80,000 spores per
syringe !!
8 mm
Exposure history of syringe after challenge
• 24 hr. room temperature, ambient pressure• Simulated flight with 7 exposures to reduced pressure • 10” Hg (334 mbar ) vacuum = 10,000 ft (3,050 m) elevation• Gas bubble expands from 3 mm to approx 4.1 mm in height
(4.5 mm theoretical)
Exposure # Duration exposure to 17” Hg vacuum
Recovery at ambient
1 2 hr 37 min 1 hr 33 min
2 2 hr 02 min 46 min
3 1 hr 35 min 2 hr 13 min
4 2 hr 07 min 15 hr 43 min
5 56 min 2 hr 10 min
6 1 hr 49 min 1 hr 24 min
7 1 hr 31 min To incubation
Results
• Incubated units at 55ºC for 48 hours
• Challenge units – 0/100 positive
• Control units– 0/100 positive
• Media placed above stopper and incubated was positive
Conclusions
• Under extreme conditions of:– Elevation simulation– Spore challenge: large challenge with unusual microbe– Number of exposures
• Mechanical movement of spores across the sterile barrier was observed
• Other areas of potential investigation– Silicon: Free vs baked on– Other lubrication systems
• Under normal shipping conditions with smaller gas bubble, contamination is unlikely
Conclusions
• Under extreme conditions of:– Reduced pressure– Spore challenge: large challenge with unusual microbe– Number of exposures
• Mechanical movement of spores across the sterile barrier was observed
• Under slightly exaggerated normal shipping conditions contamination was not observed
• Other areas of potential investigation– Silicon: Free vs baked on– Other lubrication systems
Minimizing Risk of Contamination from Stopper Movement
• Lock stopper/plunger in place with plunger rod– Syringe becomes like the vial during shipping
• Seal and sterilize the syringe in another sterile barrier (i.e pouch)– Another sterile barrier to be concerned with– Adds costs and validation
• Make the gas bubble as small as possible– Requires good control over filling/stoppering process
Stopper Movement
# of exposures to reduced pressure before sum of stopper movement exceeds Hsb
Hsb
(mm)
1 mm 2 mm 3 mm 4 mm 5 mm
4 16 8 6 4 4
5 20 10 7 5 4
6 24 12 8 6 5
7 28 14 10 7 6
Assumes:•Pressure equivalent to 8000 ft elevation•Actual movement = 70% of theoretical•Results rounded up to next whole number
Container closure integrity in pre-filled syringes vs. vials
• Standard testing method for CCI has been dye intrusion– Submerge container in a liquid containing dye– Apply vacuum and/or pressure to the chamber– Any leaks will show dye penetration into the unit
• Experiment– Placed 20 µ diameter fused silica glass capillary through
stoppers • 100 times the size of pores in a sterilizing filter!
– Assembled vials and syringes– Placed Windex solution above the stoppers
Considerations on dye intrusion CCI
• With small diameters holes in a rubber stopper, the surface tension of the dye liquid must be overcome before dye will ingress into the stopper
– Analogous to integrity testing with filters– With water based dye, a 0.22µ diameter pore in a hydrophobic
substance can require > 60 psi (>4 bar)– In a syringe, the stopper will move and decrease the pressure
differential making it more difficult to detect a leak
• In the real world, stoppers with holes will be exposed to non-sterile air not a liquid above them– Gas experiences considerably less resistance than a liquid in flowing
through a capillary– In a vial gas out will return an equal amount of gas in– Due to stopper movement, less gas will leave a syringe than in an
equivalent vial and less gas will return than left.
Dye Intrusion CCI
• Insensitive test - capable of detecting only large defects
• Less sensitive with syringes that experience stopper movement than with vials
General comments
• If a unit is non-integral during exposure to reduced pressure, gas will leave the unit.
• The amount of gas that leaves is proportional to:– The amount of the gas in the unit– The pressure differential between the inside and the outside.– The amount of gas lost and returned is less in a syringe with a
moveable stopper
• Vials typically have a much larger headspace than a syringe and therefore pose a greater risk of contamination from the returning gas.
General comments
– A vial will exchange approx. 37% of its headspace with the outside if it lacks CCI, when exposed to 8,000 of elevation
• 2 ml vial – with 1 ml fill will exchange 0.37 ml• 20 ml vial – with 10 ml will exchange 3.7 ml• A lyo cake in a 20 ml may exchange close to 7.5 ml !!!!!!
– A 1 – 3 ml syringe• 3 mm headspace will exchange less than 0.06 ml• 1 mm headspace will exchange less than 0.02 ml
Conclusions
• Stoppers in syringes can move in response to pressure changes– The amount of movement can be reduced to negligible by
reducing the gas headspace to 1 mm or less– Stopper movement helps to reduce the pressure differential
between inside and out and can reduce the amount of gas pulled back into a syringe
• A syringe is a more rugged container than a vial in the presence reduced pressure because:– Stopper movement decreases driving force– Syringes are filled with less gas headspace than a vial
THANK YOU!
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