Stopper Movement, Gas Bubbles in Shipping and Improving the Container Closure Integrity of a Pre-filled Syringe

Shawn Kinney, PhD President, 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: Hsb
HCM 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 diameter vegetative, spore smaller Approx. 60 C optimal temperature

Placing challenge solution in the syringe

Approx. 10 l 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 # 1 2 3 4 5 Duration exposure to 17 Hg vacuum 2 hr 16 min 1 hr 48 min 1 hr 47 min 2 hr 23 min 40 min Recovery at ambient 1 hr 35 min 47 min 1 hr 10 min 1 hr 36 min To incubation

Results
Incubated units at 55C for 48 hours Challenge units
14/100 positive

Control units
0/100 positive

Results
Incubated units at 55C for 48 hours Challenge units
14/100 positive

Control units
0/100 positive

Results
All positive units, presumptive ID Geobacillus Stearothermophilus

Results
Placed approx. 0.4 ml of TSB above stopper of negative challenge units All showed growth after 2 days at 55C No growth below the stopper was observed All positive units, presumptive ID: Geobacillus Stearothermophilus above 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 l 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 # 1 2 3 4 5 6 7 Duration exposure to 17 Hg vacuum 2 hr 37 min 2 hr 02 min 1 hr 35 min 2 hr 07 min 56 min 1 hr 49 min 1 hr 31 min Recovery at ambient 1 hr 33 min 46 min 2 hr 13 min 15 hr 43 min 2 hr 10 min 1 hr 24 min To incubation

Results
Incubated units at 55C 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
Assumes: Pressure equivalent to 8000 ft elevation Actual movement = 70% of theoretical Results rounded up to next whole number Hsb (mm) 4 5 6 7 1 mm 2 mm 3 mm 4 mm 5 mm

16 20 24 28

8 10 12 14

6 7 8 10

4 5 6 7

4 4 5 6

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 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 0.22 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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