LIGHTHOUSE

The Science of Pharmaceutical Manufacturing

Application Note 101

Stopper “Pop-Up” and the Effects on Container

Closure of Sterile Vials

Introduction
Container closure integrity plays an impor- Prior to sealing finished vials at the end of sec-

tant role in maintaining the stability and ondary drying, a lyophilization chamber is

sterility of lyophilized products. The in- backfilled to a gas pressure that is specified for

creased number of biological products com- the vial headspace. The specified headspace

ing to market and the need to extend product pressure varies from product to product. Typi-

shelf life has driven the growth of lyophiliza- cally, freeze dried products are stoppered at ei-

tion capacity worldwide. Lyophilization is a ther full vacuum (0 mbar absolute pressure) or

complex process that presents many manu- partial vacuum (typically 750 mbar absolute

facturing challenges one of which is main- pressure). The vacuum level serves the practical

taining and monitoring container closure purpose of helping to seat the stopper and to

integrity. Recent attention to package integ- facilitate reconstitution. Once equilibrium is

rity issues can be seen in both the revised achieved, the gas pressure in the vial headspace

aseptic processing guidance from the regula- matches the chamber pressure and the shelves

tory agencies (for example, Annex 1 revisions are lowered to seat the stoppers into the vial. At

to the European Guidelines for the Manufac- this point the vial closure integrity is estab-

ture of Sterile Products) and an increasing lished but not considered complete until the

number of package integrity related product aluminum overseal is applied. Once the shelves

recalls. Concerns over the number of custom- are raised, the seal integrity must be main-

er complaints and the cost of investigations tained for a period of time ranging from min-

and recalls has motivated investment in utes to hours to possibly days before unloading

container closure inspection systems by the and capping occur. It is possible during this

industry as a means of building quality into time for stoppers to “pop-up” allowing gas in-

manufacturing operations. gress into the vial headspace. Stoppers can pop

up due to a number of reasons including im-

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 LIGHTHOUSE
The Science of Pharmaceutical Manufacturing
Application Note 101

proper seating during shelf lowering, out of pressure rises which may impact the ability to

specification stopper and/or vial flange dimen- reconstitute the product and would likely re-

sions, and stopper coatings. sult in a customer complaint. Second, if the

product is oxygen sensitive then air ingress will

If seal integrity is lost during this time period result in oxygen exposure and potentially im-

then the physical properties of the headspace pact the product stability. Third, if container

(gas pressure and/or composition) will change closure integrity is breached then sterility can

as gas from the ambient environment outside no longer be assured.

the vial ingresses into the vial headspace. Two

common situations serve to illustrate the point. In-process monitoring systems, based on laser

If the vial is exposed to a nitrogen atmosphere, absorption spectroscopy, now exist that can

for example prior to unloading from the freeze nondestructively monitor each vial for changes

dryer, then nitrogen gas will enter the head- in the headspace gas composition and pressure.

space causing the pressure to rise. If the vial is These systems are in routine use in the industry

exposed to an air atmosphere, either because for inspecting container closure integrity in

the chamber was vented with sterile air or be- freeze dried product and, in particular, for ad-

cause the vial was exposed to room air prior to dressing the stopper pop-up issue.

capping, then air will ingress into the vial caus-

ing both the pressure and the oxygen concen- Measurement method
tration to rise. The practical implications of lost Laser absorption spectroscopy is a nondestruc-

seal integrity are threefold. First, the headspace tive method for monitoring pressure, oxygen

Container
Figure 1. Overview of nonde- (Tubing, Molded, Clear, Amber)
structive headspace mea-
surement method

Laser Detector
diode

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The Science of Pharmaceutical Manufacturing
Application Note 101

and moisture in the headspace of parenteral changes in the headspace condition of a grossly

containers. The method was first introduced to leaking vial occur within minutes. A micro-leak

the pharmaceutical industry in the late 1990’s (< 1 micron) will exhibit detectable changes in

and has been adopted and validated by compa- the headspace after a few hours to a few days de-

nies around the world for both sterile develop- pending on the initial headspace conditions.

ment and manufacturing applications. Both headspace pressure rise and oxygen in-

Container closure integrity in particular can be gress can be nondestructively monitored by la-

monitored nondestructively by headspace gas ser absorption spectroscopy.

analysis. Changes in the gas pressure or gas

composition are leak indicators. For vials stop- Systems for nondestructive headspace analy-

pered under vacuum, a leak causes a rise in sis using laser absorption spectroscopy are

headspace pressure towards atmospheric lev- configured as shown in Figure 1. Laser light

els. For vials stoppered at or near atmospheric passes through the headspace of a vial and the

pressure and exposed to air, a leak causes oxy- laser wavelength is tuned to match the absorp-

gen ingress into the vial headspace. The leak tion wavelength of oxygen at 760 nanometers

rates that result in pressure rise or oxygen in- or moisture at 1400 nanometers. Oxygen con-

gress are dependent on vial

volume and pressure differ-

Figure 2. Headspace
ential. In general the head- inspection systems incorpo-
rating the laser measure-
space pressure and oxygen
ment technology
concentration of small vol-

ume parenterals (e.g. 3-10mL)

packaged under vacuum rise

more quickly than the head-

space pressure and oxygen

concentration of large vol-

ume parenterals packaged

near atmosphere. Detectable

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The Science of Pharmaceutical Manufacturing
Application Note 101

centration is proportional to the amplitude of tions or in-process monitoring of small

the laser absorption signal and total gas pres- (<5000) batches of containers. Automated

sure is proportional to the width of the laser systems are configured for 100% inspection ap-

absorption signal. Systems are calibrated us- plications when batch sizes are large.

ing NIST traceable standards with known pres-

sure and oxygen concentration. Case study 1

In the case studies described below non-de- The first case study involves an investigation of

structive headspace gas analysis systems from container closure integrity for an oxygen sensi-

LIGHTHOUSE were used to monitor container tive lyophilized product stoppered with a ni-

closure integrity for commercial batches of ly- trogen headspace near atmospheric pressure

ophilized product. Systems, shown in Figure 2, (800 mbar). A number of vials from a com-

can be configured for manual operation or in- mercial batch showed elevated levels of oxygen

line automated operation. Manually loaded during routine QC analysis using a destructive

systems are useful for conducting investiga- oxygen analysis method. A decision was made

12 12

10 10
Headspace Oxygen (%)

Headspace Oxygen (%)

8 8

6 6

4
4
2
2
0
0
0 50 100 150 200 250 0 50 100 150 200 250
Sample # Sample #
Figure 3. Headspace oxygen content of vials located Figure 4. Headspace oxygen content of vials located
in zones 4-6. Vials from this location in the freeze in zones 1-3. Vials in these locations show air ingress
dryer showed no evidence of stopper pop up. due to stopper pop-up.

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 LIGHTHOUSE
The Science of Pharmaceutical Manufacturing
Application Note 101

to test the entire batch using nondestructive head- sure integrity of two different vial stopper combina-

space oxygen analysis. tions under actual in-process conditions. The same

A LIGHTHOUSE FMS-760 headspace oxygen analyzer vial was evaluated with a grey butyl and a teflon coat-

was used to test the entire batch for the presence of ed stopper. The lyophilized product was specified to

oxygen in the vial headspace. Vials with greater than be stoppered at 414 torr (550 mbar) of nitrogen. The

1% oxygen were to be rejected. study evaluated each vial stopper combination for

Figure 3 shows the headspace oxygen concentration its ability to hold vacuum. The study used 1000 prod-

in vials of freeze dried product that have maintained uct filled vials (500 with each type of closure) distrib-

seal integrity. The headspace oxygen levels are all be- uted over 8 shelves (4 shelves with each type of clo-

low 1%. Figure 4 shows a very different situation for a sure).

set of vials from the same batch of product. One dif- Figure 5 shows the headspace pressure in vials of

ference between the two sample sets was their physi- freeze dried product stoppered with a grey butyl

cal location inside the lyophilizer. In Figure 4 over elastomeric closure at 414 torr (550 mbar). All of

10% of the vials have lost seal integrity as evidenced by

headspace oxygen levels ranging from 1.5% to 10%. 750

It is
Nitrogen Headspace Pressure
believed that the root cause of the container closure700
Product samples using rubber butyl stoppers
integrity failures was due to stoppers not properly 650 750

700
seated when the lyophilization chamber shelves were
600

S helf 5
650
lowered. This allowed air to ingress at different rates
550 S helf 6
S helf 7
S helf 8
Pressure (Torr)

Upper S pecification (650 T orr)

500 600
resulting in oxygen concentrations over a broad T arget (41 3.7 T orr, 8 psia)
Lower S pecification (350 T orr)
S helf 5
550 S helf 6
450
range. The dependence of bad seal integrity on posi- S helf 7
S helf 8
Upper S pecification (650 T orr)
400 500 T arget (41 3.7 T orr, 8 psia)
tion in the freeze dryer allowed the manufacturer to Lower S pecification (350 T orr)

350 450
troubleshoot mechanical stoppering issues at specif- F ro nt L eft F ro nt R ight B ac k L eft B ac k R ight
C o rner C o rner C o rner C o rner
300 400
ic locations.
350
F ro nt L eft F ro nt R ight B ac k L eft B ac k R ight
C o rner C o rner C o rner C o rner
300

Case study 2 Figure 5, Headspace pressure in vials

750
sealed with
a grey butyl stopper. There is no evidence of stop-
The second case study demonstrates a process devel- 700

per pop up on any shelf or in any location on a

650 750
opment effort aimed at evaluating the container clo- given shelf.
600 700

550 650
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The Science of Pharmaceutical Manufacturing

500 600

450 550
5
400 500
 S helf 5
S helf 6
S helf 7 S helf 5
S helf 8 S helf 6
Upper S pecification (650 T orr)S helf 7
T arget (41 3.7 T orr, 8 psia) S helf 8
Lower S pecification (350 T orr)Upper S pecification (650 T orr)
T arget (41 3.7 T orr, 8 psia)
Lower S pecification (350 T orr)

c k R ight
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The Science of Pharmaceutical Manufacturing
Application Note 101
o rner
L eft B ac k R ight
ner C o rner

Nitrogen Headspace Pressure

750
Product samples using coated stoppers
700
750 The fact that bad seal integrity was not dependent on lo-

650
700
cation in the freeze dryer pointed to an issue with the

600
650
coated stopper closure system and/or an overall process
600 S helf 1
550 S helf 2 issue. Further container closure studies using nonde-
S helf 3 S helf 1
550 S helf 4 S helf 2
500 Upper S pecification (650 T orr)S helf 3 structive headspace analysis allowed the manufacturer
T arget (41 3.7 T orr, 8 psia) S helf 4
500 L ower S pecification (350 T orr)Upper S pecification (650 T orr)
450 to optimize the stoppering process. Closure failures
T arget (41 3.7 T orr, 8 psia)
L ower S pecification (350 T orr)
450
400
with the coated stopper were lowered from > 15% of the
400
350
F ront Left F ront R ight Back L eft Back R ight
batch to < 1% with the optimized process. A headspace
C orner 350 C orner C orner C orner
300 F ront Left
C orner
F ront R ight
C orner
Back L eft
C orner
Back R ight
C orner
inspection machine was implemented for 100% final
300

Figure 6, Headspace pressure in vials sealed product inspection guaranteeing the detection and re-
with a teflon coated stopper. There is widespread
jection of any residual vials that had lost container clo-
evidence of stopper pop up on all shelves and in
all locations on any given shelf. sure integrity.

these vials have maintained seal integrity. The Conclusion

headspace pressure levels are uniform and match Stopper pop-up resulting in loss of container closure in-

the pressure set in the lyophilzation chamber pri- tegrity is not uncommon and impacts the stability, ste-

or to lowering the shelves. Figure 6 shows the rility and reconstitution of lyophilized product. Non-de-

headspace pressure in vials from the same batch structive headspace analysis is a powerful method for

that were stoppered using a teflon coated elasto- monitoring container closure integrity in finished vials

mer closure. Over 15% of these vials did not main- of freeze dried product and for building quality into the

tain seal integrity after removal from the lyophili- manufacturing operation. Monitoring absolute pres-
zation chamber. The gas ingress into these vials sure changes and/or oxygen ingress in the vial head-
resulted in headspace pressures from 30 to 300 space serve as leak indicators. Manually loaded systems
torr (40 to 400 mbar) above the target level. are valuable tools for small scale studies and investiga-

tions, and fully automated systems have been imple-

mented and validated for 100% product inspection in

commercial scale applications.

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