Enzyme Cocktails & Bio-Stabilisation on Biosensors

Applied Enzyme Technology Ltd.

Guido Drago

Outline of Presentation

• Why do we need stabilisation of proteins?• Choice of stabilisers• Influencing factors• Examples of stabilised proteinsExamples of stabilised proteins• Protein immobilisation and stabilisation• AET ProductsAET Products

Proteins Stabilised by AET Ltd.

21 proteins stabilised in the dry state28 proteins stabilised in solution

Horseradish Peroxidase-Conjugated AntibodiesAlkaline Phosphatase conjugated antibodies

Esterases HydrolasesEsterases HydrolasesKinases LipasesLuciferase OxidasesOxidoreductases PeroxidasesOxidoreductases PeroxidasesPhosphatases Proteases

Most contract research has led to the generation of stable enzyme formulations from between 50 days to over 18 months at temperatures of up to 50°C

Why Do we need Stability?

• All products require both shelf life and operational stability

Shelf Life

All products require both shelf life and operational stability

• Most biosensors require at least 6 months shelf life, in fact most specifications require between 1-2 years storage stabilityp q y g y

• If a sensor cannot be stored without refrigeration for extended periods that sensor will never become a viable product

Why Do we need Stability?

Operational Stability

• Operational stability is dependant on the type of biosensor.

Operational Stability

• Disposable sensors can be active from seconds to several minutes. Reusable sensors may require several days to several

th t bilit ( bl l )months stability (reusable glucose sensor)

• The stability of a sensor may be the difference between a research prototype and a commercial sensorprototype and a commercial sensor

• The stability specification must be determined early on in developmentdevelopment

Why Do we need Stability?

Solution Stability

Solution stability is required during the manufacturing process of the biosensor

Wh th th i l id d b i ti i d ttiWhether the sensor is laid down by screen printing or via dotting proteins can be extremely labile in solution for extended periods.

The drying process i e extraction of the moisture from the enzymeThe drying process, i.e. extraction of the moisture from the enzyme solution on the sensor surface, is probably the major process step which will lead to the inactivation of the majority of proteins.

Shelf lifeShelf lifeOperational stabilitySolution stabilitySolution stability

Via the introduction of the rightVia the introduction of the right stabiliser formulation and protocol all of these stability issues can be solved!these stability issues can be solved!

We can help!!We can help!!

Stability Troubleshooting

• Stabiliser pre-screening is carried out using optical methodologyStabiliser pre screening is carried out using optical methodologyand or electrophoretic techniques, these can interrogate large numbers of stabiliser combinations.

• Choice of stabilisers, buffer, pH, ionic strength, protein concentration are critical.Choice of stabilisers, buffer, pH, ionic strength, protein concentration are critical.

• Dry Formulation (air dry, vaccum dry, freeze dry). Dependant on sample volumes and equipment available.

• Store at elevated temperature in order to increase speed of degradation (preferred temperature 37°C).

• Ensure storage in low humidity and low oxygen environment.Ensure storage in low humidity and low oxygen environment.

• Measure enzyme activity regularly.

• Transfer enzyme/ stabiliser formulation to biosensor for further investigation.Transfer enzyme/ stabiliser formulation to biosensor for further investigation.

Choice of Stabiliser

• Determine the isoelectric point of your protein

Thi ill bl t id tif th t f l l t l t th t ill bi d•This will enable you to identify the type of polyeletrolytes that will bind your protein of interest

• If you are unable to do this you can either use gel electrophoresis to y y g pdetermine the binding avidity of the protein/polyelectrolyte complexes

• Or use a small panel of positively, negatively or neutrally charged polyeletrolytespolyeletrolytes

• The recommended polyols are less easy to determine. However there are a small number of standard molecules which are quickly screened

Enzyme Stabilisation: Influencing factors

The single most important parameter in ti f t t l t bilit f t i ipromotion of structural stability of proteins is

retaining the surface water activity of the protein. By adding polyhydric alcohols the p y g p y ysurface water activity is modified relative to the absolute concentration of the additive.

Enzyme Stabilisation: Influencing factors

When used in combination with additivesWhen used in combination with additives promoting electrostatic interactions (polyelectrolytes) or surface chemical interactions leading to immobilisation or crosslinking, the efficacy is usually enhanced significantly, indicating synergy of action at the surface of theindicating synergy of action at the surface of the protein structure thereby stabilising the protein overall.

Enzyme Stabilisation: Influencing factors

Some additives such as metal ions are directly related to enzyme structure and asdirectly related to enzyme structure and as such are not strictly surface interactions. Addition of dilute solutions of metal salts

i l i ft t bilieg. magnesium, calcium often stabilise proteins to a high degree and act synergistically with polyelectrolyte combinations.

The Mechanism of Stabilisation

1 Polyalcohols

• The polyalcohols include sugars and sugar alcohols

1. Polyalcohols

• Polyalcohols modify the water environment surrounding a protein thus replacing and competing for free water within the systemsystem

• This modified hydration shell confers protection to the protein maintaining 3D structure and biological activityg g y

• This enables storage of biological materials both in solution and in the dehydrated state

The Mechanism of Stabilisation

2 Polyelectrolytes

• Polyelectrolytes include numerous polymers of varying charge and structure

2. Polyelectrolytes

structure

• The interactions between proteins and polymers are electrostatic

• These form large protein-polyelectrolyte complexes which retain full biological activity

• At AET we have the ability to calculate the stoichiometry of protein/polymer binding which we have determined as critical for maintaining enzyme activity in stabiliser formulations

The Mechanism of Stabilisation

3 St bili C bi ti

• Where polyelectrolytes and polyalcohols are combined a synergistic effect is usually observed

3. Stabiliser Combinations

synergistic effect is usually observed

• Ratios of polyelectrolyte to polyalcohol are extremely important in the overall stabilisation of proteinsimportant in the overall stabilisation of proteins

• The buffer type, pH, ionic strength, concentration and ratio of stabilisers to protein/enzyme all play crucial roles in protein p y p y pstabilisation both in the dry state and in solution

The Mechanism of Stabilisation

The addition of polyelectrolytes to solutions of proteins promotes the formation of soluble protein/polyelectrolyte complexes by electrostatic interaction. Polyhydroxyl compounds are then able to penetrate the

t t ff ti l l di t t bili tistructure more effectively leading to stabilisation

The Detection of Protein/polymer Complexes by Isoelectric Focusing

+

y g

_

1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 12

1= Markers 7=L - GLDH+ 0.5%Polymer 3-

2=L -GLDH 8=L - GLDH+ 0.5%Polymer 4

3=L GLDH+ 0 5% Polymer 1 9=L GLDH3=L -GLDH+ 0.5% Polymer 1 9=L - GLDH

4=L -GLDH+ 0.5% Polymer 2 10=L -GLDH + 0.5% polymer 5

5=L -GLDH 11=L -GLDH

6=Markers 12=Markers

The Detection of the Stoichiometry of Polymer Binding to the Thermophilic Form of GLDH

A B

p

+ +A B

GLDH

GLDH-PEComplex

- -

p

Free enzyme detected(0.01% Polymer 1)

Free enzyme detected(0.01% Polymer 2)

Alcohol Oxidase

AOX Hansenula Polimorpha and Pichia Pastoris - Stability trials - 6 Months%

100

120

140

%

60

80

100

0

20

40

-20

0Time 0 1 Month 2 months 3 months 4 months 5 months 6 months

Aox Pichia Pastoris Control AOX Pichia PastorisST 05 Aox Hansenula Polimorpha Control Aox Hansenula Polimorfa ST 06

Alcohol Oxidase stability as determined by microtitre plate assay

Glucose Dehydrogenase

GDH NAD Dependant and PQQ Dependant - Stability trials

200

250

%

100

150

50

100

0Time 0 Time 1 1 w 2 w 3 w 1 Month

GDH NAD Dependant Cont GDH NAD Dependant P 48 GDH PQQ Dependant - CONT GDH PQQ Dependant P51

Glucose dehydrogenase stability as determined by microtitre plate assay

Printed Electrode with Mediator: P t S S fProcesses at Sensor Surface

FAD + ( )

FADH2 ( d)

GlucoseMed (ox)Med (red)2e- GOX

(ox)(red)(ox)(red)

Glucose Biosensor

Calibration Graphs Over Time (sol 2 500mV)

0 0000070.0000080.000009

0.00001e

Am

ps

0 0000010.0000020.0000030.0000040.0000050.0000060.000007

Ave

rage

resp

onse

09/12/200416/12/200419/01/200501/02/2005

00.000001

0 2 4 6 8 10 12 14

Glucose Concentration

A

Glucose oxidase biodotting solution stored for extendedGlucose oxidase biodotting solution stored for extended time prior to biosensor construction and testing

The PolyEnz Process

This process produces pre-stabilised enzymes which can then be immobilised onto or into a multitude of matricesbe immobilised onto or into a multitude of matrices.

This methodology can be applied to:• The adhesion and stabilisation of biomolecules to biosensor surfaces• The production of stabilised biocatalysts involved in the biotransformationof targets for the Pharmaceutical & Chemical Industries

The adhesion and stabilisation of biomolecules to biosensor surfaces

• The stabilisation of enzymes & microbial targets used in

This process has been demonstrated on solid activated particulate supports

• The stabilisation of enzymes & microbial targets used in bioremediation

supports

Significant improvements in the shelf-life of the immobilised biocatalyst have been recorded

The PolyEnz Process

• The PolyEnz Process changes the microenvironment of the immobilised biocatalyst, which can lead to higher enzyme activity levels

• Improved production process longer shelf life and operational stability• Improved production process, longer shelf life and operational stability

• Improved process efficiency and reduced production costs

Acetyl Choline Esterase

Dry Stability of AChE B03

80

100

120

140

%

0

20

40

60

-1 24 49 74 99 124 149 174 199 224 249 274 299

Activ

ity -

%

NH2COOH

NH2COOH

Protein

-20

Time at 37°C - days

Derivatised PolymerAcetyl choline esterase stability determined by microtitre plate assay

Carbon Electrode

Conclusions

•Stabilisation of enzymes is crucial for the production of a commercially viable biosensor.

•The choice of stabilisers can be quickly determined by optical assays.q y y p y

•Forming cross-linkable complexes in the presence of stabilisers can be used to manufacture stable disposable and reusable sensors.

•Determining the pI of your protein, optimal pH, buffer type and ionic strength of the environment required all go a long way to stabilising your protein of interest.

•The introduction of cofactors and metal ions into your stabilisationformulation will also enhance specific enzymes significantly.

•Maintaining a low water and oxygen environment during the storage of the complete biosensor is essential for long term stability

Protein Stabilisation Kits

AET manufactures 5 kits

Biosensor Trouble shooting Kit

Contents:

Stabiliser Formulations

Pre-stabilised EnzymesPre-stabilised Enzymes

Surfactant

Complete biodotting formulation

Array of screen printed sensor designsUsing different electrochemical materials

El t h i l t lElectrochemical protocols

Handi-Lab: Novel Technology for the Field Measurement of Ammonia

The Handi-Lab delivers a low skill, low cost, fast quantitative measurement of ammonia in water samples.

One shot disposable sensors.Integrated sampling device.Recyclable materials.

Digital output within 5 minutes.No pre-calibration required.Measurement range 1-10 parts per million of ammonia

Recyclable materials.

Measurement range 1-10 parts per million of ammonia.Automatic adjustment for sample and ambient temperature.Fully portable and battery operated.On board data storage and down loading facility.Measures within 10% bias 10% errorMeasures within 10% bias 10% error.Allows on site testing and ‘live’ mapping of pollution events.Biosensors stable for at least 6 months at room temperature .

Electrode Manufacture & Enzyme Deposition

Screen Printed Electrodes

GEM Clean Room Printing Facility AET Biodot Facility

The Gwent GroupThe Gwent Group

• Can provide a unique range ofCan provide a unique range of materials and services for all biosensor systemsbiosensor systems

• GEM produce materials and base pelectrochemical transducers

• AET provide biostabilisation services and biosensor fabrication

Applied Enzyme Technology LtdApplied Enzyme Technology Ltd.

• Monmouth House • Mamhilad Park • Pontypool • Torfaen • NP4 0HZ • United Kingdom • Tel: 00 44 (0) 1495 750505 • Fax: 00 44 (0) 1495 752121• Fax: 00 44 (0) 1495 752121 • Email: sales@gwent.org• Website : http:www.gwent.orgWebsite : http:www.gwent.org