Producing Handling and Storing Heat Sensitive Proteins in Middle East

8/13/2019 Producing Handling and Storing Heat Sensitive Proteins in Middle East

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Debayan Ghosh

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EPYGEN IS A BIOTECHNOLOGY

RESEARCH AND MANUFACTURINGORGANIZATION

Epygen Vision is to create a world class Life Science

organization which will emphasise on strong research

combined with an applied focus on Industrial, Pharmaceutical

and Agriculture Biotechnology.

The company explicitly fosters individual creativity and

initiative among employees, encouraging scientists

and technologists to keep product pipeline full with productsand solutions for tomorrow.





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Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Average

Maximum

Temperature C

24.0 25.4 28.2 32.9 37.6 39.5 40.8 41.3 38.9 35.4 30.5 26.2

Average MinimumTemperature C 14.3 15.4 17.6 20.8 24.6 27.2 29.9 30.2 27.5 23.9 19.9 16.3

Mean Rainfall

(mm)

15.6 25.0 21.0 7.0 0.4 0.0 0.8 0.0 0.0 1.2 2.7 14.9

Mean No. of Days

with Rain

5.4 4.9 5.9 2.6 0.3 0.0 0.5 0.6 0.1 0.2 1.3 3.6

Sunshine Hours /

day

8.2 8.5 8.6 10.2 11.3 11.5 10.7 10.5 10.4 9.9 9.3 8.2

Mean Sea

Temperature C

20.9 20.7 22.3 25.0 28.5 31.2 32.2 32.9 31.9 29.9 27.0 23.4

Dubai Temperature Chart

Source : Met Department Web Site

For Enzyme Protein: Months To Watch: May-October



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CLIMATE CHALLENGE FOR A BIOTECH

COMPANY:

1.HOW TO MAKE PRODUCTS THAT ARE

MORE STABLE.

2.HOW TO MAKE STABLE FORMULATIONS.





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Denaturation of Proteins means...

Disruption and destruction of both the secondary and tertiary

structures.

Since often denaturation reactions are not strong enough to

break the peptide bonds, the primary structure (sequence of

amino acids) remains the same after a denaturation process.

Denaturation disrupts the normal alpha-helix and beta sheetsin a protein and uncoils it into a random shape.

Something like this..





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Native and denatured states

native statefolded state

denatured ensembleunfolded ensemble

single structure or ensemble

of very similar structures;

compact

many different structures fluctuating;

not usually very compact;

disordered but not a random coil

For some proteins, but not all, this process is readily reversible and occurs

without populated intermediate forms--> two-state folding





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Heat energy disrupts hydrogen bonds and non-polar hydrophobic interactions as it increases thekinetic energy and causes the molecules to vibrateso rapidly and violently that the bonds aredisrupted.

Actually what happens when

protein feels too hot..

As temperature is increased, a number of bonds in the protein molecule are

weakened.

The first affected are the long range interactions that are necessary for the

presence of tertiary structure.

As these bonds are first weakened and are broken, the protein obtains a more

flexible structure and the groups are further exposed.





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Theoretically, even if heating ceases at this stage the

protein should be able to readily refold to the native structure

(renaturation) and you are lucky.....

As heating continues, some of the cooperative hydrogen bonds

that stabilize helical structure will begin to break.....

As these bonds are broken, water can interact with and form

new hydrogen bonds with the amide nitrogen and carbonyl

oxygen of the peptide bonds.





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The presence of water further

weakens nearby hydrogen bonds by

causing an increase in the effective

dielectric constant near them.

As the helical structure is broken,

hydrophobic groups are further

exposed.........




Cli k H & U d


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The effect of exposure of new hydrogenbonding groups and of hydrophobic groups is to

increase the amount of water bound by the proteinmolecules.

The unfolding that occurs increase the hydrodynamic radius of themolecule causing the viscosity of the solution to increase.

The net result will be an attempt by the protein to minimize its free energy by burying

as many hydrophobic groups while exposing as many polar groups as possible to the

solvent.

While this is analogous to what occurred when the protein folded originally, it is

happening at a much higher temperature.

This greatly weakens the short range interaction that initially direct protein folding

and the structures that occur will often be vastly different from the native protein.






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Naive view of fo ld ing

thermodynamics

Native

(folded)

Denatured

(unfolded)

Gu

Gu = Hu - TSu

++

unfolded state

more disordered

favorable native state

interactions broken

Gu

T

Hu

TSu

protein becomes less stable at

high temp and unfolds when TS

exceeds H

0






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Amount of unfo lded

prote in as func t ion of T

Gu = Hu,Tm(1 T/ Tm )

+ Cp[T T

mT ln (T/ T

m)]

Ku = exp(Gu/RT) = [U]/[F]

fu = Ku / (1 + Ku )

fraction unfolded

concentration unfoldedand folded

Keq

for unfolding reaction

eqn describing Guas function of T

set of nested equations






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Drawbacks of Chemical processes:

Many chemical processes inherent drawbacks from

commercial and environmental point

Non-specific reactions result in poor product yields

High process temperatures and pressure required

High energy costs

Often causes devastating environmental implications.

These drawbacks can be eliminated by using

enzymatic conversions.






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Enzyme reactions

Usually require mild conditions;

Highly specific,

Involve very fast reaction rates

Carried out by numerous enzymes playing

different roles in an orchestrated manner.

Robust scope of kinetics

BUT OFTEN ENZYMES ARE

EXPOSED TO HARSH PROCESS

HEAT !!!!!!!!






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Examples

1.90+ Degrees

Centigrade..AMYLASE in

Desizing.

2.80+ Degrees Centigrade..Xylanase

for Feed Pelletization.

3.100+ DegreesCentigrade.Cellulases for Petroleum

Industry Downhole action.






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Why?

A. Higher expression

B. Higher purity (%protein:other junk)C. cheap

D. can engineer protein

E. can express enzymes which are found

in pathogenic organisms

Heterologous Protein Expression

Homologous Protein Expression

WHY Most industrial enzymes are

produced Recombinantly


C c e e & Upg ade




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BY PROTEIN ENGINEERING

TOOLS.

To Render

Enzymes

more

Thermostable


pg




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After mutating a Glucoamylase Enzyme from Aspergillus species the thermal unfolding of

the catalytic domain , 13

-helices obeyed the random ordered mechanism.....

In which the -helices 8, 1 and 11 unfolded more rapidly than the others.

The catalytic center was well protected by ()6-barrel at simulation temperatures up to

600 K,.....

The catalytic base, E400, migrated from its original interior pocket to the surface of thecatalytic domain by jumping the hydrophobic barrier provided by -helices 12 and 13 at

800 K.

Improving Thermostablity bystructurally reinforcing or locking

-Helix by disulfide links.

Ref: Hsuan-Liang Liu, Wen-Chi Wang(2003), Protein Eng., 16, 19-25

What was happening in the GlucoamylaseProtein

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When a disulfide bond is

engineered to lock the -helix 11

on the surface of the catalytic

domain, it was observed todramatically increase the

Thermostability......


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This indicates that the introducedresidues with higher hydrophobicitywere favorable in the loop between-helices 12 and 13, whereas theypartially destroyed the hydrogenbond and salt linkage network in the

catalytic centre.....-Helices 12 and 13 can bestabilized by introducing residueswith higher hydrophobicity......

This was an example how proteincan be stabilized by proteinengineering.


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E d d F t


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MESOPHILES versus EXTREMOPHILES:

Mesophiles 35-60oC

pH 4-8

Extremophiles organisms that colonize with

one or more extreme environmental

parameters

pH

temp

salinity

pressure

LOOKING INTO NATURE


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E d d F t


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HYPERTHERMOPHILES;

90oC plus

typically anaerobic

found at tectonically active sites

vents

springs

geysers


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Hyperthermophiles:

Difficult to culture

Once has to move to

recombinant production oftheir enzymes

Purification easier

Just Raise temp..

But often


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Polyol-induced water-mediatedeffects such as an increase in thesurface tension of water play a majorrole in the stabilization of proteins

Preferential hydration of proteinsobserved in Polyol presence is aconsequence of the increase in thesurface free energy of water.

Stabilizing Proteins in

formulation with Polyols


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We believe that thermodynamicmeasurements coupled with biologicalactivity might be a valuable tool forscreening additives in liquid proteinformulations for protection against variousdegradation mechanisms causing protein

conformational destabilization associatedwith loss of (or decline in) biologicalactivity.

How We Measure Thermal

Stability in our Dubai Plant


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No single polyol could provide maximalprotection against all protein

destabilization routes.

Therefore, polyols should be selectedon the basis of possible destabilization

mechanism for a particular proteinlikely to be encountered duringformulation, process development,handling or storing.

Which Polyol To

chose?


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Control 73.7 0.6 5.9 0.5 29.0 1.9

Mannitol 75.9 0.8 7.3 0.6 36.0 0.8

Sucrose 75.3 0.4 6.8 0.4 33.0 0.6

Lactose 75.2 0.2 6.7 0.3 33.0 0.4

Glycerol 74.9 0.4 6.2 0.8 30.0 1.2

Propylene

glycol 71.4 0.5 5.4 0.3 27.0 1.2

Polyols Transition Tem Calorimetric Enthalpy Specific Enzyme

Cal/mol X 104 Activity X 103

Effect of Polyols on Midpoint Transition

Temperature, Calorimetric Enthalpy, and SpecificEnzyme Activ-ity of Lysozyme Subjected toAggregation/Precipitation by Sodium Chloride

Source: Somnath Singh, Jagjit Singh, 2003, AAPS PharmSciTech ; 4 (3) Article 42


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Assessment of

Denaturation

Ultraviolet Adsorption Spectroscopy.

This measures the wavelength and amount of ultraviolet radiation absorbed by a

molecule.

In proteins, both the wavelength and extent of absorption depend on the amino acids

present and on their physical environments. Giving a fare idea on changes the proteinHas gone through...


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The interaction of polarized light with protein can be

measured by the techniques of circular dichroism and

optical rotatory dispersion.

These methods yield an indication of the extent of repeating

structures present in protein and are generally utilized to

give estimates of the amount of secondary structure present,

eg. alpha-helix, beat sheet or coil.

Circular dichroism

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For those proteins like our ENZYMES, denaturation can be defined as the loss of

structure to render the enzyme inactive.

Changes in the rate of the reaction, the affinity for substrate, pH optimum, temperatur

optimum, specificity of reaction, etc., may be affected by denaturation of enzyme

molecules.

Loss of enzymatic activity can be a very sensitive measure of denaturation as some

assay procedures are capable of detecting very low levels of product.

We are CONCERNED

about the Loss ofBiological Activity



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This assay measures the release of reducing sugars by the action of acellulase on a cellulosic substrate.

One unit of activity liberates 1 micromole of reducing sugar

(expressed as glucose equivalents ) in one minute under the conditionsdescribed. The sample activity is then related to a standard with astated CMCase activity.

EPYGEN CMC-DNS-ASSAY


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Accelerated

Stability and

Normal

stability at

Dubai

Temperatures

after POLYOL

stabilization

Days October to April

Normal Stability

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Dubai Enzyme Company perspective.

1.Liquid Enzymes are more in danger during summer months.

2.Between Production and formulation, unstable enzyme shouldbe stored in refrigerated conditions.

3.Immediately after production, move drums in cold room.

4.Prefer cold distribution and reefer containers during July toSeptember.

5.Protein Salting out is first sign to watch for.

6.Check turbidity and consistence

7.Maintain Accelerated and Normal stability data for all newformulations.8.Compulsory First In First Out inventory.

9.Container to carry temperature rea-ltime logging.


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Producing Handling and Storing Heat Sensitive Proteins in Middle East

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