Detection of microorganisms in
pharmaceutical samples using
synthetic polymer foot printing
‘MICROPRINT’
28th August 2015
Theme 1
Eithne Dempsey, Santhosh Padmanabhan and Brian Seddon
Niall O’Reilly and Marc Kelly
Knowledge Day – 28th August 2015
Objective Theme 1
Demonstration of a selective electronic microbiological assay methodology for
implementation of bacteria detection in pharmaceutical ingredients.
ITT Dublin – assay development, electronic profiling of redox molecules
WIT – generation of bacterial imprinted polymer films “artificial receptors”
“rapid, simple, cost-effective test for detecting microbial contamination
as an alternative to current slow or expensive techniques –
an early warning system”
Knowledge Day – 28th August 2015
MICROPRINT Technology
Cell imprinted polymers for selective capture of bacteria
Quantification based on electronic enzyme profiles
(A) Cell Imprinted Polymers
BIO-CARD &
Hand-held meter
(B) Electrochemical Assay Development
Knowledge Day – 28th August 2015
• Integrated microfluidics
• Cell imprinted polymer for
selective capture
• Electronic sensors-cellular
enzyme profiles
Summary USR – Industry Input
Specification
Combined Industry Response
Current method to
improve
Total viable count,
Aseptic manufacturing environments
Species
Wide variation – E. coli as proof of principle
Sample type
Environmental monitoring, water samples,
product and raw material, aq. based
Sample size
1-200 mL
Time to result
< 4 hours or same day test
Sensitivity
1 cfu/100 mL – 1cfu/mL
Test type
Fully automated, single use disposable
Sample throughput
20 samples/day
Knowledge Day – 28th August 2015
Current Technologies
TVC Bioburden
ChemScan® RDI (Fluorescent labeling - solid phase cytometry)
BactiFlow ALS (Fluorescent labeling – flow cytometry)
D-Count (Fluorescent labeling - flow cytometry)
PTS-Micro™ (Fluorescent labeling - solid phase cytometry)
BIOTRAK- Real time viable particle counter for air samples (Fluorescence)
Microbe Identification
BiOLOG (redox chemistry)
VITEK® (VITEK 2 & VITEK-MS - metabolic based assay)
MicroSEQ® (Genotype based assay)
Endotoxins
Endosafe®-PTS (Limulus Amebocyte Lysate (LAL) assay)
• Incubation time before analysis: * 20 – 48 hours
• Analysis time : 4 – 5 hours
*depends on application
Knowledge Day – 28th August 2015
State of the Art
• Chemscan RDI
–
–
–
–
–
Sensitive; 1 cfu/100ml in single shift (4-6 hrs)
Cost per sample €75 + labour
Capital cost €200,000
Trained scientist and sample preparation
No speciation, requires organism identification
• MALDI-TOF
–
–
–
–
–
1 day analysis
Cost per sample ~ €100 + labour
Capital cost > €200,000
Trained scientist (complicated)
Limited to known database, requires additional PCR
Knowledge Day – 28th August 2015
Performance vs.
Current Technologies
• Plate counting (most common method)
–
–
–
–
MICROPRINT Technology
3 to 5 days
Multi-step aseptic prep
Requires training
Sensitive (1 cfu/ml)
• Optical / cytometry methods
–
–
–
–
–
Up to 18 hours (for 1 cfu/ml)
Multi-step operation
Requires training
Up to €100 per sample
Sensitive
(dependant upon application)
Knowledge Day – 28th August 2015
7 hours
Single-step on-chip
No training required
< €1 per sample
Sensitive 1 cfu/100ml
MICROPRINT Technology
offers….
Operation
Step 1: Sample (100 mL) is injected through on-card filter/fluidic and incubated at 37C for signal amplification.
Step 2: Following on-cartridge incubation (1-7 h), the BIO-CARD is then inserted into a handheld reader
Step 3: Results are displayed within minutes and the cell count is displayed in the handheld meter.
MICROPRINT Technology USP
•
•
•
•
•
•
•
•
•
No user training required
No sample preparation
Cost effective (reader < €500 sample < €1)
Self-contained (no external reagents required)
Capable of multiplexed operation
Results within same shift (< 8 hrs)
Facilitate rapid go/no-go decisions within manufacturing environment
Sensitive - 1 cfu/100ml
Non-destructive to enable bacterial recovery and serotyping
Knowledge Day – 28th August 2015
Value Proposition for
Pharma Industry
• Raw material testing
• In-process testing
• Microbial limit testing
• Bioburden assessment
• Process water testing
• Environmental monitoring
• Sterility testing
• Personal hygiene
• Root-cause analysis (identification of contaminant)
Knowledge Day – 28th August 2015
Overview of Progress
Three strands to date….
(A) E. coli

An electrochemical bio-assay protocol based on enzyme profile biochemistry developed and
standardized for the quantification of E. coli
Escherichia coli

Rapid detection of E. coli down to 1 cfu/100 mL achieved (time to result 7 h)

Designed and engineered BIO-CARD for E. coli detection – testing underway

Assessment of E. coli imprinted polymers – evaluated the performance of imprinted polymer
membranes from WIT
(B) Staphylococci & Micrococci spp. (Aseptic pharma manufacturing)

Electrochemical assay developed for simultaneous detection and identification of
Staphylococci & Micrococci spp.

Rapid detection of cells down to 102 cfu/mL achieved (time to result 10 min)
(C) Total Viable Aerobic Count

Electrochemical assay developed for the quantification of total viable aerobic bacteria
Knowledge Day – 28th August 2015
Detection of E. coli
Electrochemical Assay development: E. coli - β-galactosidase
Enzyme reaction
Chronocoulometry Measurements: E1: -0.2 V; E2: +0.2 V
E. Coli /
Electrochemical detection of p-aminophenol
*Values are the meanSD of triplicate measurements
n=50 over 5 day study
Substrate: p-Aminophenyl β-D Galactopyranoside
Inducer: IPTG
Incubation temp. : 37oC; Incubation time: 7 h
Knowledge Day – 28th August 2015
MICROPRINT BIO-CARD
MICROPRINT BIO-CARD fabrication sequence
Electrochemical detection of
p-aminophenol @
MICROPRINT BIO-CARD
Knowledge Day – 28th August 2015
Commercial Opportunity - Aseptic
Manufacturing in Pharma Industries
Transfer of contamination
Species from the genus Staphylococci contribute to 38.4% of total positive
samples while Micrococci species contributes to 22.4%.
Current technologies – Visual test; qualitative
Catalase test
Oxidase test
Catalase
Oxidase
+
+
+
Staphylococci
Micrococci
+ve
Bubbles resulting from
production of O2 gas clearly
indicate a catalase +ve.
-ve
Microdase Discs - qualitative
procedures to aid in the
differentiation of staphylococcus
from micrococcus by the
detection of the oxidase enzyme.
• Incubation time before analysis: 18 – 20 hours
• Analysis time : 10 min
Focus is to transfer the current visual test to a rapid electrochemical detection system
BIOCARD for the simultaneous detection and identification of staphylococci & micrococci spp.
Knowledge Day – 28th August 2015
Electrochemical Assay
Development
Catalase test
The enzyme catalase decomposes hydrogen peroxide into
water and oxygen
2 H2O2  2 H2O + O2
Oxidase test
Reaction of tetra-methyl-p-phenylenediamine (TMPD)
with oxidase systems
Knowledge Day – 28th August 2015
Total Viable Count
 Total Viable Aerobic Count (TVC) indicates the presence
of microorganisms such as bacteria and fungi (yeast & moulds) in
samples.
 Count represents the number of cfu/g (or) cfu/mL of the sample.
CO2 is a universal metabolite produced by all aerobic microorganisms.
Indicator organisms
Bacteria
Yeast
•
E. coli
•
•
Staphylococcus aureus
Mould
•
Pseudomonas aeruginosa
•
•
Salmonella spp
Candida albicans
Aspergillus niger
Knowledge Day – 28th August 2015
Electrochemical CO2 Sensor
Electrochemical CO2 sensor will utilize the acidic nature of CO2 for detection. It consists of a gas-permeable membrane in which a
pH-sensitive redox molecule is immobilized together with a buffer.
CO2 permeating into the membrane changes the internal pH of the buffer. With in turn changes the redox behavior of the molecule.
CO2 dissolves in water it exists in chemical equilibrium producing carbonic acid
CO2 + H2O
CO2 (g)
CO2 (dissolved) with
H2CO3
Carbonic acid
where kH=29.76 atm/(mol/L) at 25 °C
pCO2 (atm)
pH
[CO2] (mol/L)
[H2CO3] (mol/L)
[HCO3−] (mol/L)
[CO32−] (mol/L)
10−8
7.00
3.36 × 10−10
5.71 × 10−13
1.42 × 10−9
7.90 × 10−13
10−7
6.94
3.36 × 10−9
5.71 × 10−12
5.90 × 10−9
1.90 × 10−12
10−6
6.81
3.36 × 10−8
5.71 × 10−11
9.16 × 10−8
3.30 × 10−11
10−5
6.42
3.36 × 10−7
5.71 × 10−10
3.78 × 10−7
4.53 × 10−11
10−4
5.92
3.36 × 10−6
5.71 × 10−9
1.19 × 10−6
5.57 × 10−11
10−3
5.42
3.36 × 10−5
5.71 × 10−8
3.78 × 10−6
5.61 × 10−11
10−2
4.92
3.36 × 10−4
5.71 × 10−7
1.19 × 10−5
5.61 × 10−11
10−1
4.42
3.36 × 10−3
5.71 × 10−6
3.78 × 10−5
5.61 × 10−11
100
3.92
3.36 × 10−2
5.71 × 10−5
1.20 × 10−4
5.61 × 10−11
101
3.42
3.36 × 10−1
5.71 × 10−4
3.78 × 10−4
5.61 × 10−11
Knowledge Day – 28th August 2015
Redox pH sensor
−21.8 mV/pH (28oC)
@ SPE
vs. Ag/AgCl
More acidic
Mcilvaine’s Buffer system (0.2 M Na2HPO4 and 0.1 M citric acid)
Knowledge Day – 28th August 2015
TVC measurement –
electrochemical CO2 sensor
Single colony of
Staphylococcus aureus
in CASO
Staphylococcus aureus
in CASO
Overnight culture
(Incubation @ 37oC / 200 rpm)
105 cfu/mL
Staphylococcus aureus
in CASO
Serial dilution
Staphylococcus aureus
Control (with no cells)
Casein-peptone soymeal-peptone broth
Knowledge Day – 28th August 2015
Overall to date …
MICROPRINT E. Coli detection
“presence or absence”
Overall to date
Sample Volume : 100 mL
Cells detectable : 1 cfu
Detection time : 7 h
cfu/100mL
107
106
105
104
103
102
10
1
Time, h
0:10
1:00
2:00
3:30
4:30
5:30
6:30
7:00
Knowledge Day – 28th August 2015
Technology Roadmap
MICROPRINT
• Handheld
• €1/sample
• Minimal user training
• Sample prep. on-chip
• 1 cfu/mL
Knowledge Day – 28th August 2015
Cell Imprinting Protocol
• Simple and cost efficient synthesis.
• Robust organic polymeric materials resistant to
extreme pHs and temperatures.
• Reusability in excess of 100 extraction cycles.
• Versatility; can be applied in a variety of formats
Imprint
Polymer Coating
Knowledge Day – 28th August 2015
E. coli model system
Significant response from E. coli on MIP compared to NIP and P.
aeruginosa controls
Knowledge Day – 28th August 2015
Extension of technology
Significant response from P. aeruginosa on MIP compared to NIP and E.
coli controls
Knowledge Day – 28th August 2015
Current Work Packages
• WP 9 Optimisation of Cell-Imprinting technology for
integration into Bio-Card
• WP 10 Development of stand-alone Leptospira detection
kit
• WP 11 Full electrochemical profiling of E. coli cellimprinted polymer
• WP 12 Generation of cell-imprinted polymers for the
capture of pharmaceutically relevant microorganisms
Knowledge Day – 28th August 2015
WP 9 – Optimisation of
Imprinting
• Processing parameters for film deposition analysed, spray
coating and drop-casting not optimal
Knowledge Day – 28th August 2015
WP 9 – Optimisation of
Imprinting
• Capture of lower cell-density on optimal imprinted surface
ascertained
Knowledge Day – 28th August 2015
Imprint Selectivity
• Competition binding in the presence of microbial mixtures
Knowledge Day – 28th August 2015
WP11 – Electrochemical
Profiling
• Integration of imprint and electrochemical detection – utilises
flow cell to model fluid dynamics of potential bio-card
• Initial results incredibly promising
Knowledge Day – 28th August 2015
WP11 – Electrochemical
Profiling
• Initial results incredibly promising
• Performance variation posing real issue – particularly recent
samples
• All efforts are focused on rectifying this issue
Knowledge Day – 28th August 2015
WP10 Leptospira Detection
• Fastidious (no standard USP test) pathogen, passes through
0.22µm filters.
Knowledge Day – 28th August 2015
WP12 Relevant Organisms
• Wide range of species procured (including fungal, staph,
micrococci)
• MIPs prepared for E. coli, P. aeruginosa, MRSA
• Further imprints planned but
not reliant upon QCM
Knowledge Day – 28th August 2015
Current Status
• Smart polymer capable of selective capture of target microbes
developed and optimised
– E. coli model integrated into electrochemical flow-cell
• Amenable to range of targets even complicated fastidious
organisms
• Integration with electrochemical assay for Bio-Card promising
yet in need of further work
Knowledge Day – 28th August 2015
Future Work
• Development of BIO-CARD for the detection of E. coli and
performance evaluation
• Development of BIO-CARD for TVC quantification based on
CO2 sensor
• Parallel development of CATOXE system
•
Optimisation of imprinting protocol and performance evaluation
– selectivity studies and BIOCARD adaptation and testing
•
Develop multiple organism specific cards
– each requires validation
•
Stand alone testing kit of complex organisms
Knowledge Day – 28th August 2015