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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

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



5 40 min To incubation

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


2 2 hr 02 min 46 min


4 2 hr 07 min 15 hr 43 min

5 56 min 2 hr 10 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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