Assessing the Cleanliness of Medical Devices
Stephen Spiegelberg
Presented at the 3rd Annual Orthopaedic Manufacturing &
Technology Exposition and Conference (OMTEC 2007), June
20-21, 2007, Chicago, IL.
Cambridge
Polymer Group, Inc.
Testing, Consultation, and Instrumentation for Polymeric Materials
Background of Cambridge Polymer Group
• Contract research company formed in 1996
• Core competencies
– polymer R&D
– test methodology development
– instrumentation design
• Biomedical community
–
–
–
–
–
–
–
bone cement
crosslinked polyethylene
spine
eye
vascular
product recalls
cleanliness of medical devices
Sterile is not the same as Clean
• Sterile: live microorganisms content is below acceptable
levels
– Bacteria, yeast, fungi, molds, viruses
– Sterility Assurance Limit (SAL): probability that an implant will
remain nonsterile following sterilization
• 10-6 (one in a million)
• Clean: non-live residue content is below acceptable
levels
– Pyrogens – dead but deadly
– Chemicals
– Particulate matter
Sterile is not the same as clean
Methods of Sterilization
Methods of Cleaning
Glutaraldehyde
Detergent Wash
Ethylene oxide
Alcohol Wash
Steam/Dry Heat*
Acid Passivation
Plasma
Air blasting
Ionizing radiation*
High pressure rinses
Sonication
*issues with polymeric devices
Case Study I
• In 2000, Sulzer Orthopedics noticed higher
than normal revision surgeries on their
InterOp Acetabular Shell
• High failure rate in isolated manufacturing
group
Explanted hip components
showed little tissue ingrowth
into the porous titanium
backing, even after 11
months of in vivo use.
Spiegelberg, Deluzio, Muratoglu, Trans Orthopedic Research Society, 2003
InterOp Acetabular Shell
Cementless fixation: relies on osseointegration in porous titanium structure
What Happened?
Independent Research Team
Pathologists
Manufacturing Experts
Analytical Labs
Believed to be related to a manufacturing residue
ƒ
Try to identify type of residue in order to determine best analytical
technique
ƒ
Design sample preparation procedure to extract and quantify
residue
ƒ
Validate extraction and analysis technique
ƒ Determine resolution levels
Preliminary Information
• Suspected that a residue was on implants
• Introduction believed to be from machining lubricants
• Received sample
lubricants from
manufacturer
Protocols
• Extract residue from component
– solvent selection
• Analyze mass of residue with quantified
technique
• Identify composition
• Look for trends with manufacturing
Solvent Selection
Select solvent that does not have an IR absorption band in the region of the target material
Good solvent for target material
5
oil
Carbon tetrachloride
Chloroform
4
H
H
absorbance [A.U.]
oil
3
2
Cl
1
Cl
C
Cl
Cl
0
Carbon tetrachloride
-1
3900
3400
2900
2400
1900
-1
w avenumber [cm ]
1400
900
400
Extraction Protocol
Sonicate for 1 hour
Carbon tetrachloride
Rinse component
Concentrate solution
(77 C)
Protocol on web site: www.campoly.com
Infra-red Spectroscopy
C
H
Transmission T = I / I 0
Absorbance A = log10 (1/ T )
Beer’s Law: A = αbc
α = absorptivity of chemical species
b = path length of cell
c = concentration of chemical species
Used to identify hydrocarbon-based components in residue
Calibration Curve for Oil
70
Oils A, I, K, F, D
A = αbc
1 mm path length
Areas baseline-corrected
60
A 2819-2992
cm-1
50
A = 325.27c
40
2
R = 0.9967
30
20
10
0
0
0.05
0.1
0.15
hydrocarbon concentration [wt.%]
0.2
0.25
Validation Study
Oil oil
content
[mg]
content [mg]
Porous titanium test samples coated with known amounts of oil
45
45
40
40
35
35
30
30
Oil Measurement Validation Study
Applied Oil
Me a s u re d O il C o n te n t
25
25
20
20
15
15
10
10
5
5
0
0
1
1
2
2
3
3
4
4
5
5
6
6
7
7
sample
Corrections for hydrocarbons in solvent
8
8
9
9
10 1 0
Oil in Shelf-Stored Components
• Examined oil content in over 500
acetabular shells
• Grouped component lots by
manufacturing history
Manufacturing Procedure
Group
1
Machine
titanium
shell
Apply
porous
coating
Detergent
wash&rinse
High temperature
sintering
Nitric acid
passivation
Group
2
Peg chamfer
Group
3
ID turn
Group
4
No passivation
Manufacturing History Analysis
80
70
Oil Content (mg)
60
Group1
Group2
Group3
Group4
88% of failures
50
passivation
no passivation
40
30
20
10
0
1260000
1280000
1300000
1320000
1340000
1360000
1380000
1400000
1420000
1440000
1460000
Lot Number
No statistical dependence of oil content on manufacturing history
Oil Removal by Nitric Acid Passivation
1 hour soak in 27 vol.% nitric acid
70
A Series
K Series
AIK Series
Residual Oil Content [mg]
60
50
40
30
20
10
0
Control
Passivation Only
• Nitric acid passivation does not remove measurable quantities of oil
Fluid Absorption Analysis
• CsTi test coupons weighed, then soaked in water/bovine
serum
• Determine fluid absorption by weight difference
• Dependence on presence of oil, nitric acid passivation
Fluid Absorption Analysis
0.3
0.3
0.25
0.25
0.2
0.2
0.15
0.15
0.1
0.1
Red symbols: no passivation
Blue symbols: nitric acid passivation
0.05
0.05
0
0
20
40
total fluid uptake [ml]
fluid absorption [g]
Mineral Oil A
60
80
100
0
120
oil content [mg]
Presence of oil affects fluid absorption only through volumetric means
No major effects of nitric acid observed
Histopathology of Tissue from 113 InterOp Shells
• Acute and chronic inflammation in periprosthetic tissue, with an
abundance of lymphocytes, granulation tissue, neutrophils, and
giant cells. Staining was highly positive for IL-1b and Il-6 activity
[1].
• Inflammation was found in the capsule as well, and was not
therefore relegated to tissue in direct contact with the device.
• Concluded that a substance in the oil, rather than the oil itself,
was responsible for the inflammation [1, 2].
1. Campbell, P.M., J; Catelas, I. Examination of Recalled Inter-Op Acetabular Cups for Cause of Failure. in Society for Biomaterials. 2002. Tampa, FL.
2. Campbell, P.M., J; Catelas, I. Histopathology of tissues from Inter-Op acetabular sockets. in 48th Annual Meeting of the Orthopaedic Research Society. 2002.
3 Week Rabbit Study
Tissue response in rabbits
injected with Oil I
Tissue response in InterOp
patients
No
Yes
Chronic Inflammation
82.1%
Extensive
Eosinophils
96.4%
Minimal
Giant Cells
14.3%
Abundant
No
Yes
Acute Inflammation
Fibrunous Exudate
Lipogranuloma
82.1%
No
Only 2 pathological markers were shared in the two studies
Granulation Tissue
No
Abundant
Lipid Droplets
46.4%
No
No
Yes
Other Foreign Body
3.6%
Yes
Fibrous Tissue
42.9%
Yes
Necrosis
3.6%
Yes
Metal
Bloebaum, R.D., E.L. Whitaker, J. Szakacs, and A. Hofmann. The tissue response to an injection of gamma sterilized mineral oil in rabbits. in 49th Annual Meeting of
the Orthopaedic Research Society. 2003. New Orleans, LA.
Nitric Acid + Oil
0.8
0.7
residue-top layer (primarily oil)
residue-bottom layer (primarily acid)
oil A
0.6
absorbance [A.U.]
0.5
removed
0.4
0.3
0.2
removed
0.1
0
new
-0.1
2000
1900
1800
1700
1600
1500
1400
1300
1200
w ave num be r [cm -1 ]
• There is a modest chemical change in the oil
with exposure to acid
• GC/MS analysis on residues was inconclusive
•Cytotoxicity testing on the residues came back
negative
1100
1000
Could Endotoxins be the culprit?
• Histopathology of endotoxins produced a similar tissue response as
that observed in the Inter-Op tissue [1].
• Nitric acid passivation can reduce the levels of endotoxins adhered
to titanium samples [2].
• Endotoxins were found in the sump water of the machine shop
• Trace amounts could be stationed at the oil-tissue interface, enough
to prevent osseointegration
a lipopolysaccharide (LPS) produced from Gram-negative bacteria
1. Greenfield, E.M., Y. Bi, A.A. Ragab, V.M. Goldberg, J.L. Nalepka, and J.M. Seabold, Does endotoxin contribute to aseptic loosening of orthopedic implants? J. Biomed. Mater Res,
Part B: Appl. Biomater., 2005. 72B: p. 179-185.
2. Merritt, K., S.A. Brown, and V.M. Hitchins. Ability of nitric acid or acetone to inactivate bacterial lipopolysaccharide (LPS). in 28th Annual Meeting Transactions of the Society for
Biomaterials. 2002
Conclusion of Case I Study
• Oil present on all manufactured lots tested, including
those with successful outcomes
– Specific manufacturing history associated with failed
implants
• Explanation of clinical response
– not related to absolute level of oil
– appears to be related to nitric acid passivation step
• Most likely culprit was an adherent endotoxin that
was delivered via the oil, and was not inactivated by
nitric acid passivation
Case Study II: Residue Identification
•
What manufacturing residues are introduced?
– Release agents
– Lubricants/coolants
– Polishing compounds
– Cleaning agents
– Particulate debris
– Eluants from packaging
– Chemicals from gloves
•
Identification allows the manufacturer to identify and control the
source of the residue production
Further Identification of Residues
Analytical techniques
– FTIR indicates quantity of oil, but not identity
• Sum of all parts
– Soluble residue: Gas Chromatography/Mass
Spectroscopy
– Insoluble residue: Energy dispersive
spectroscopy (EDS)
Gas Chromatography/Mass Spec.
GC Phase: Separates the compounds
injected grouped chemical
chemicals are separated
carrier gas
to mass spec
liquid phase
• species partially dissolves in liquid phase
• re-enters gas stream when partial pressure in gas
stream drops
Mass Spectrometer
Identifies the compounds
analyzer
ionizer
heated filament
e-
detector
+
+
+
from GC
+
Calibrate detector with metallic salts
ion current
207Pb
203Ti
205Ti
208Pb
200
212
mass/charge
•
•
•
•
Ionizer: Electrons create ions from gas molecules
Analyzer: Filters by mass-to-charge ratio (m/z)
Detector: Measures ion current, proportional to quantity of single
m/z molecules
Good up to around 400-500 g/mole
GC/MS Analysis of Residues
• Catalogued all compounds that may have come into
contact with machined components
• Identified which residue composition was present on
component
• Identify what manufacturing step introduces residue
Roughing Pump Oil
chemical composition
octadecane
nonadecane
1-hentetracontanol
1-chloro-octadecane
17-pentatriacontene
(1-butylhecadecyl)-cyclohexane
1-octadecanethiol
(2-hexyloctyl)-cyclopentane
N-methyl-N-[4-[4-methoxyl-1-hexahydropridyl]-2-butynyl]-acetamide
1',4 dihydroxy-2,3' dimethyl-, (-)-[1,2'-binaphthalene]-5,5',8,8'-tetrone
1-docosene
*GC/FID: approx. 1-10 ppm
approximate
concentration %
1
1
1
11
17
19
6
9
7
25
3
Insoluble Residue
• Scanning Electron Microscopy
• Energy Dispersive X-ray Spectroscopy (EDS
or EDXA)
– Qualitative/semi-quantitative
– particulate matter adhered to carbon tape,
then analyzed in Phillips XL-30 FEG SEM.
EDS System (in-line with SEM)
X-ray tube
sample
detector
Incident electron
or X-ray
ejected electron
K
X-ray
K
L
M
L
M
Energy of X-ray {KeV]
0.1-1 wt.% resolution
EDS Analysis of Extracted Residue
aluminum
EDS Analysis of Extracted Residue
10 microns
titanium
silica
iron
vanadium
sodium
Conclusions to Case II
• Manufacturers have techniques to
identify type of residue
• Source of residue in manufacturing
process
Case Study III: Residue on Explants
• Can we extract and identify manufacturing
residues on explanted devices?
• Possibly identify source of implant failure
Explant Analysis
•
•
Problem: biological tissue are
hydrocarbon-based
H
O
H
H
H C
O C
C
C H
H
triglyceride
Look very similar to hydrocarbon-based
manufacturing residues under FTIR
H C
O
H
O C
C
O
H
H
O C
C
4
3.5
3
oil
lipids
H-C Stretch
H C
H
H
10
H
H
C H
10
H
H
C H
10
H
absorbance [A.U.]
2.5
mineral
oil
1.5
1
0.5
0
-0.5
-1
3400
H
H H
2
3300
3200
3100
3000
2900
2800
-1
wavenum ber [cm ]
2700
2600
2500
2400
C H
H C C
H H
10
H
Explant Analysis
• Remove lipids from solution
• Protocol similar to shelf-stored
analysis
– Sonication with carbon tetrachloride
– Evaporation
– Filtration
• Isolates manufacturing hydrocarbons
from lipids and most other biological
residues
– infra-red analysis
Explant Analysis
Pre-doped devices extracted in presence of 5 grams of acetabular tissue
90
Applied amount
80
Measured amount
no filtration
oil content [mg]
70
60
50
40
30
20
10
0
shell 1
shell 2
Cadaver Analysis
• Shells were doped with known amounts of oil, then placed in cadavers for 24
hours
• Extraction, then filtration
Cadaver Analysis
Cadaver Specimens
45
40
oil
oil content
content [mg]
[mg]
35
ir-measurement
4500
4000
ir-measurement
Applied oil
Applied
residue oil
content-gravimetric
residue content-gravimetric
4000
3500
35
3500
30
30
3000
3000
25
25
2500
2500
20
20
2000
2000
15
15
1500
1500
10
1000
10
1000
5
500
5
0
500
0
az197
az198
az199
az200
az201
0
0
az197
az198
az199
az200
az201
residue mass
mass [mg]
[mg]
45
40
4500
Identification of Extracted Residue from Explant
• FTIR provides limited information on structure of
hydrocarbons
– Data provided from labs that show hydrocarbons on all explants
(3000 cm-1)
– Immediately conclude that these are manufacturing residues
– Presence of carbonyls indicate these are probably fatty acids (1700
cm-1)
– Systemic long-chain hydrocarbons(1,2)
• GC/MS identifies specific composition of hydrocarbons
1. Liber, A.F. and H.G. Rose, Saturated hydrocarbons in follicular lipidosis of the spleen. Arch. Path., 1967. 83: p. 116-122.
2. Rose, H.G. and A.F. Liber, Accumulation of saturated hydrocarbons in human spleens. J. Lab. & Clin. Med., 1966. 68(3): p. 475-483.
GC/MS on Explant Residues
Fenclofenac: non-steroidal antiinflammatory drug
Tranylcypromine: is a non-hydrazine
monoamine oxidase inhibitor, an
antidepressant and antimanic (brand
name Parnate)
Nitrazepam: sleep
medication
Tricosene
Elemental Analysis of Explants
• Manufacturing residue and tissue analysis
directly on explant
• Scanning electron microscopy/Energy
dispersive spectroscopy
• No damage to explant (non-destructive)
Elemental Analysis of Explants
Case III: Explant Analysis Summary
• Validated techniques to quantify manufacturing
hydrocarbon content
• Residue composition can be analyzed with GC/MS
• Surface residue can be analyzed directly on devices
• Tissue composition can be identified (elements)
Case Study IV: Other Sources of Residues
• Manufacturer trying to identify all possible
source of possible contamination on
components
• Determine residues from
– gloves
– packaging
Gloves Analysis
•
Gloves were sonicated in carbon tetrachloride, then analyzed
with GC/MS
Extracted residue: 190 mg
Vinyl gloves
component
phthalic anhydride
bis (2-ethylhexyl) phthalate
1,2-benxenedicarboxylic acid, diisononyl ester
didecyl phthalate
1,2-benxenedicarboxylic acid, isodecyl octyl ester
1,2-benxenedicarboxylic acid, bis(1-methylheptyl) ester
1,2-benxenedicarboxylic acid, diheptyl ester
1,2-benxenedicarboxylic acid, diisodecyl ester
approximate
concentration %
1
80
5
4
5
3
1
1
Extracted residue: 30 mg
Latex Gloves
component
pentachloro-cyclopropane
benzothiazole
N,N-dibutyl-formamide
butylated hydrotoluene
tetraphenyl-pyrazine
trichloro-methanesulfonyl chloride
acetic acid, octadecyl ester
hexatriacontane
tetracosane
heneicosane
dotriacontane
approximate
concentration %
3
9
9
6
15
3
24
6
12
6
6
Packaging Analysis
• Examine extractables from biomedical component
packaging
• Chemical extractables
– solution extraction/FTIR
• Debris generation
– Scanning electron microscopy
Chemical Extractables from Packaging
• GC/MS of residue extracted from a component
– Phthalate (plasticizer for PET packaging)
– Residue from foam packaging
1.8
foam residue
1.6
dimethyl ester, dodecanedioic
1.4
absorbance [A.U.]
1.2
1
0.8
0.6
0.4
0.2
0
3900
3400
2900
2400
1900
-1
w ave num be r [cm ]
1400
900
400
Packaging Debris Analysis
Electron micrograph of plastic pouch used to hold metallic implant
How Clean is Clean Enough?
• Determined by:
– Analytical detection limits
– Achievable with commercial cleaning
processes
– Cost of cleaning
• Not possible to remove all residue
– Levels that do not elicit an adverse
biological response
• Application area in the body
• Size of device
ASTM Activities
• ASTM activities on cleanliness issues of
medical devices (F04.15.17)
– Passed first standard on assessing cleanliness
(ASTM F2459)
– WK15532: Guide for Assessment of
Contamination and Residues on Medical devices
• Compilation of known assays for residues, including
endotoxins
– WK13292: Standard Practice/Guide for Shipping
Possibly Infectious Materials, Tissues, and Fluids
Conclusions
• Acceptable levels?
Good historical success of medical device
= acceptable levels of residues
Acknowledgements
•
•
•
•
Dr. Gavin Braithwaite
Dr. Orhun Muratoglu
Denise Saltojanes
Partial funding from Sulzer Orthopedics
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