Specialty products I:

Biopharmaceuticals

Wouter Hinrichs

Pharmaceutical Technology and Biopharmacy

University of Groningen
The Netherlands

Email: W.L.J.Hinrichs@rug.nl
 Content
2

Introduction therapeutic proteins

Instability therapeutic proteins
Stabilization of therapeutic proteins
Drying methods: - Freeze drying
- Spray drying
- Spray freeze drying
 3

Biopharmaceuticals
- Elucidation human genome
- Recombinant DNA technologies
- Developments biotechnology

Therapeutics
 4

Examples biopharmaceuticals:
- Proteins
- Peptides
- Nucleic acids (pDNA, siRNA)
- Vaccinesa
-

a) Vaccines can be proteins, peptides, oligosaccharides, nucleic acids but also virus like particles
 Examples therapeutic proteins
5

- Enzymes: a-glucosidase (myozyme) Pompes disease

- Monoclonal antibodies: bevacizumab colon cancer

- Cytokines: interferones, interleukines various

- Hormones: insuline diabetes

- Subunit vaccines: haemagglutinin/neuroamidase flu

 Examples of marketed products
6

Protein drugs approved by the United States Food and Drug Administration (FDA) in 2015

Protein Trade name Type

Alrocumab Praluent Monoclonal antibody
Asfotase alfa Strensiq Enzyme
Daratumumab Darzalex Monoclonal antibody
Dinutuximab Unituxin Monoclonal antibody
Elotuzumab Empliciti Monoclonal antibody
Evolocumab Repatha Monoclonal antibody
Idarucizumab Praxbind Monoclonal antibody
Insulin degludec Tresiba Hormone
Mepolizumab Nucala Monoclonal antibody
Necitumumab Portrazza Monoclonal antibody
Parathyroid hormone Natpara Hormone
Sebelipase alfa Kanuma Enzyme
Secukinumab Cosentyx Monoclonal antibody
 Structure proteins
7

Proteins are built up from amino acids

- Primary structure:
Sequence amino acids

- Secondary structure:
Specific three-dimensional structure elements,
e.g. -sheets and a-helices

- Tertiary structure:
Relative orientation structure elements

- Quaternary structure:
Relative orientation various polypeptide chains
 Amino acids
8

http://www.personal.psu.edu/staff/m/b/mbt102/bisci4online/chemistry/chemistry8.htm (Downloaded on 15-12-2015)

 Primary structure
9

R R
H2N - C - CO2H + H2N - C - CO2H
H H
- H2O

R O H R
H2N - C - C - N - C - CO2H
H H
N-terminal C-terminal
 Primary structure
10

R O H R R O H R
H2N - C - C - N - C - CO2H H2N - C - C - N - C - CO2H
H H H H

For example:

H O H CH3 H 3C O H R
H2N - C - C - N - C - CO2H H2N - C - C - N - C - CO2H
H H H H
Glycine - Analine Analine - Glycine
 Secondary structure
11

http://www.abcte.org/files/previews/biology/BioMod%203%5B1%5D.3%20secondary%20structure.jpg (Downloaded 15-12-2015)

 Tertiary structure
12

Open loops

Alpha helix

Beta pleated
sheets

http://click4biology.info/c4b/7/pro7.5.htm (Downloaded on 15-12-1015)

 Quaternary structure
13

Structure of bovine Lglutamic dehydrogenase

A globular protein consisting of six homologous monomers

http://www.rcsb.org/pdb/images/3mvo_bio_r_500.jpg?bioNum=1.
 Overview structures
14

http://www.slideshare.net/nayeemdaisu/3-protein-structure-30449199 (Downloaded on 20-1-2016)

 Classification
15

Simple:
- Composed of amino acid residues only

Conjugated:
- Also contains e.g. metal ions, carbohydrates (glycoproteins),

Hemoglobin
http://themedicalbiochemistrypage.org/protein-structure.php#quaternary
 Peptide - protein?
16

1. Peptides do not have a tertiary structure

while proteins do

2. Polypeptides having < 40 amino acids: peptides

Polypeptides having > 40 amino acids : proteins
 How many different proteins
can theoretically exist?
17

- Example: protein composed of 100 amino acids

(averaged sized protein)
- 20 different amino acids exist

20100 different proteins are theoretically possible

This is: 10130 or a one followed by 130 zeros
 The problem
18

The action of a protein is determined by its 3-D structure

The 3-D structure is maintained by relatively weak interactions:
- Hydrogen bonds
- Van der Waals forces
- Hydrophobic interactions
- Ionic interactions
- Disulfide bridges

Note: all interactions are non-covalent

except for disulfide bridges

http://www.uic.edu/classes/bios/bios100/lectures/chemistry.htm
 Cold chain
19

Proteins are produced as aqueous solutions

In an aqueous environment, proteins are generally unstable
Proteins solutions should be stored and transported refrigerated,
i.e. 2-8 oC (cold chain) but even then their shelf life can be limited

In particular a problem in tropical developing countries

- Expensive
- Health care workers often lack sufficient knowledge of
cold chain management
Cold chain
20
 Cold chain
21

Transport in remote areas

The solution?
22
 23

Degradation mechanisms

Chemical degradation: Physical degradation:

Deamidation Aggregation
Oxidation Gelation
Hydrolysis Denaturation
Disulfide exchange Adsorption
... ...
 An example
24

Degradation pathways of oxytocin

LC/MS

After thermal stress:

13 degradation products could be identified!
Most degradation products associated with disulfide bridge

Avanti C et al, AAPS J, 13 (2011) 284-290

Avanti C et al, Mol Pharm, 9 (2012) 554-562
 Specific approach
liquid formulations
25

Oxytocin solutions in 5 mM citrate buffer pH 4.5 and various

concentrations divalent cations stored for 4 weeks at 55C

Stabilization by specific interaction between oxytocin, citrate

buffer and divalent cations protecting the disulfide bridge

Avanti C et al, AAPS J, 13 (2011) 284-290

 General approach
liquid formulations
26

Extremolytes
Organic osmolytes from extremophilic microorganisms

Accumulated in response to environmental changes

- Increased or decreased temperature
- Drastic changes in salt concentrations

Stabilization of biological macromolecules

 Examples extremolytes
27

O HN
+
O
HO OK N
H
O O
HO
O
Ectoine
OH Trehalose
HO OH
HN
OH + CH 3 O
O +
N
Firoin H H 3C N C O
O H2
CH 3
Hydroxyectoine
Betaine
 Mechanism
28

Preferential Exclusion Theory

Sf Sf*

Entropy
Sfu
Su
Sfu*
Solvent
Su*
Solvent + Stabilizer

Sfu* > Sfu

 Another general approach
liquid formulations
29

In-vitro:
- Steric stabilization by PEGylation
Covalent linkage of poly (ethylene glycol) chains to the protein

Particle can not approach each other by:

- Osmotic effect
- Entropic effect

Disadvantage: PEGylation may affect activity of the protein

 Effect PEGylation
in vivo behavior
30

- Particles are not recognized by the immune system

Long circulating particles or Stealth particles
Passive or active (homing device) targeting

homing device

- Increased size
Less clearance by the kidney (if peptide / small protein)
(cut-off value kidneys: 20 kDa)
 Alternative strategy
31

Most degradation reactions require molecular mobility

Reduce molecular mobility

Bring protein in the dry state

Concomitant advantage:
- Development of various non-parenteral dosage forms is possible
(e.g. tablets for GI tract delivery or powders for pulmonary delivery)
 Drying techniques
32

- Freeze drying
- Spray drying
- Spray freeze drying
- Vacuum drying
- Foam drying
- Super critical fluid drying
-
 Drying techniques
33
Aqueous protein
solution

N2(l)

Spray drying (Spray-)freeze drying

 Drying stresses
34

Freeze drying: freezing and dehydration stresses

Spray drying: shear, thermal, and dehydration stresses
Spray freeze drying: shear, freezing and dehydration stresses

Drying of proteins without protection causes damage

Stabilizing excipient required

Literature: sugars
 Mechanisms
35

Mechanisms of stabilization by sugars

Water replacement

Particle isolation

Vitrification / shielding
 States of matter (1)
36

Thermodynamically stable
(equilibrium states)
Crystala:
- Molecules arranged in a lattice
- Low translational mobility of the molecules

T > Melting temperature (Tm)

Liquid:
- Random orientation of the molecules
- High translational mobility of the molecules

a): if polymorphism, only one of the crystalline forms is thermodynamically stable

 States of matter (2)
37

Thermodynamically unstable
(non-equilibrium states)
Glass:
- Random orientation of the molecules (amorphous)
- Low translational mobility of the molecules
- Kinetically stable: crystallization cannot occur

T > Glass transition temperature (Tg; always: Tg << Tm)

Rubber:
- Random orientation of the molecules (amorphous)
- High translational mobility of the molecules
- Kinetically unstable: crystallization can occur
- Super-cooled liquid
 States of matter (3)
38

Vapor
Tv
Temperature

Liquid
Tm

Crystal Rubber
(Supercooled liquid)

Tg
Glass
(Amorphous)
 Phases/states
39

Liquid Rubber

Crystal Glass
 The glassy state
40

- Viscosity: 1012 - 1014 Pa.s (water: 10-3 Pa.s)

- Material does not flow

- Material is able to carry its own weight

- Viscosity rapidly decreases over and above
the glass transition
 Mechanisms
41

Mechanisms of stabilization by sugars

Water replacement

Particle isolation

Vitrification / shielding
 Water replacement
42

H-bonds between biopharmaceutical and H2O are gradually replaced by H-bonds

between biopharmaceutical and sugar-OH
Sugar molecules should be oriented towards the irregular surface of the protein
Sugar must be in the amorphous state
 Particle isolation
43

Aggregation or intermolecular Aggregation or intermolecular

reactions are possible reactions are NOT possible

- Sugar matrix acts as a physical barrier

between the particles
- Sugar should have low translational mobility
 Vitrification / shielding
44

Formation of a protective sugar coating around the protein

Drug is shielded from the environment (oxygen/light)
The sugar matrix should form a tight coating in which diffusion on a
realistic time scale is inhibited (compare fly in amber in the movie
Jurassic Park)
Sugar must not only be in the amorphous state (tight coating)
but also in the glassy state (no diffusion)
 The glassy state
45

The glassy state is not only required for the bulk and vitrification/shielding
mechanism but also for the water replacement mechanism!
Amorphous state is required, but rubbery state is not stable and
crystallization can occur.

Effects crystallization:
- Loss of H-bonding
- Mechanical forces during crystallization may damage the protein
 Freeze drying
46

Phase diagram of water

Pressure (atm)

Critical point
Liquid
Solid
(ice)
0.006 Triple point

Gas (vapor)
0.01
Temperature (oC)
Sublimation curve
 Phase diagram binair/
condensed system
47

x-axis: composition
y-axis: temperature
Temperature

Composition:
via lever rule

Relative Relative
amount of B amount of A

100% A x 100% B
 Phase diagram water/sugar
48
Temperature

Solution Sugar
crystals
+
Ice + Solution Solution

Ice + Sugar crystals

Pure water Pure sugar

The vapor phase is ignored; pressure is fixed

 Phase diagram water/sugar
49

Cooling of a homogeneous solution with Cooling of a homogeneous solution with

composition W(T0) from To T1 results in composition W(T0) from To T2 results in
phase separation: ice and a homogeneous phase separation: ice and a homogeneous
solution with composition W(T1) solution with composition W(T2)

T0 T0

Temperature
Temperature

T1 T1

T2 T2

Pure W(T0) W(T1) Pure Pure W(T0) W(T2) Pure

water sugar water sugar
 Phase diagram water/sugar
50

Cooling of a homogeneous solution with Cooling of a homogeneous solution with

composition W(T3) from To T2 results in composition W(T0) from To T3 results in
phase separation: crystalline sugar and a phase separation: ice and crystalline sugar
Homogeneous solution of composition W(T4)

T0 T0

Temperature
Temperature

T1 T1

T2 T2

T3 T3
Pure W(T3) Pure Pure W(T0)
Pure
water W(T4) sugar water sugar
 Phase diagram water/sugar
51

Temperature
Tm,sugar

Homogeneous
Crystallization solution Crystallization
curve of water curve of sugar

Tm,water (273 K)
Te Two phases:
sugar crystals and
aqueous sugar
Two phases: solution
Two phases: Ice + Sugar Crystals
ice and aqueous
sugar solution Pure water Pure sugar

Only thermodynamic stable (equilibrium) states

 State diagram
52

Also thermodynamic unstable (non-equilibrium) states

Temperature
Crystallization Tm,sugar
curve of sugar

Crystallization Homogeneous solution Rubber

curve of water
Tg, pure sugar

Tm,water (273 K)
Tg
Glass transition
curve

137 K Ice + Glass (Cg)

Pure water Pure sugar
Cg

(Pure glassy water can only be obtained when the cooling rate is > 3,000,000 K/s)
 The glass transition curve
53

Temperature

Glass transition curve

Pure water Pure sugar

w 1 Tg ,1 k w 2 Tg , 2
Gordon-Taylor equation: Tg (mix)
w1 k w 2
 Gordon-Taylor equation
54

w 1 Tg ,1 k w 2 Tg , 2
Gordon-Taylor equation: Tg (mix)
w1 k w 2
What is k?

Many approaches:

Tg ,1 1 a 2 Cp , 2
k k k
Tg , 2 2 a 1 C p ,1
 Gordon-Taylor equation
55

Tg ,1 1
Simha-Boyer rule: k
Tg , 2 2
1 and 2 are density of component 1 and 2, respectively
Tg,1
When 1 = 2 k
Tg,2
w 1 Tg ,1 k w 2 Tg , 2
Subsitute in: Tg (mix) and rearrange
w1 k w 2
(see next slide)
1 w1 w 2
Fox equation:
Tg (mix) Tg,1 Tg,2
 Fox equation
56

Tg ,1
w1 Tg ,1 w 2 Tg , 2
Tg , 2 w1 Tg ,1 Tg , 2 w 2 Tg ,1 Tg , 2
Tg (mix )
Tg ,1 w1 Tg , 2 Tg ,1 w 2
w1 w2
Tg , 2
( w1 w 2 ) Tg ,1 Tg , 2 Tg ,1 Tg , 2

w1 Tg , 2 w 2 Tg ,1 w1 Tg , 2 w 2 Tg ,1

1 w1 Tg , 2 w 2 Tg ,1 w1 Tg , 2 w 2 Tg ,1 w1 w 2

Tg (mix ) Tg ,1 Tg , 2 Tg ,1 Tg , 2 Tg ,1 Tg , 2 Tg ,1 Tg ,1
 Gordon-Taylor equation
57

w 1 Tg ,1 k w 2 Tg , 2
Gordon-Taylor equation: Tg (mix)
w1 k w 2

But, k is usually considered as a fitting parameter

Three component systems:

w1 Tg ,1 k 1, 2 w 2 Tg , 2 k 1, 3 w 3 Tg , 3
Tg (mix)
w 1 k 1, 2 w 2 k 1, 3 w 3
Freeze driers
58
 Freeze dried product
59

Highly porous cake

Reconstitution is quick, no or only minimal agitation is required
 Spray drying
60

Procedure:
- Solution is aerolized using a nozzle
- The aerosol is exposed to hot air (evaporation is endothermic temperature
in the droplets is much lower than the environmental temperature )
- Dry powder particles are collected

Solution Compressed air

Also other types of nozzles are available

 Freeze drying
61

Phase diagram of water

Pressure (atm)

Critical point
Liquid
Solid Evaporation
(ice) curve
0.006 Triple point

Gas (vapor)
0.01
Temperature (oC)
 Spray drying
62

Tm,sugar

Temperature
Homogeneous Tg, pure sugar
solution

A B
Tm,water (273 K)

137 K

Pure water Pure sugar

Spray driers
63
Spray driers
64
Spray dried product
65
 Spray freeze drying
66

Procedure:
- Solution is atomized by a nozzle
- Droplets are collected in liquid nitrogen (-196 oC) or on a cold surface
- Frozen droplets are freeze dried
Spray freeze dried product
67
 Which type of sugar?
68

High glass transition temperature (Tg)

High Tg of the maximally freeze concentrated fraction (Tg)
High sugar content of the maximally freeze concentrated
fraction (Cg)
Slow crystallization
No reducing groups (Maillard reaction)
Low hygroscopicity
Flexible molecular structure (if oligo- or polysaccharide)
Non-toxic
Cheap
Readily available
 Maillard-reaction
69

Sugars with aldehyde group: Amadori reaction

NR
H O
C H C
+ H2NR - H2O
H C OH H C OH

R R
Cross-linked protein
Sugars with ketone group: Heynes reaction aggregates (browning)
H H

H C OH H C OH
+ H2NR - H2O
C O C NR

R R

Incompatibility reducing groups (aldehyde of ketone) of

sugars with amine groups of proteins
 Maillard-reaction
70

-H2O
aldose sugar N-Substituted ketosylamine
[ketose sugar] [aldosylamine]
+H2O
Amadori rearrangement
[Heyns rearrangement]
1-amino-1-deoxy-2-ketose
[2-amino-2-deoxyaldose]
-2H2O high pH
-3H2O
low pH high
temperature Strecker
reductones
+R-NH2 degradation

-2H +2H +R-NH2

fission products
furfural dehydro-
(carbonyls, dicarbonyls aldehyde + CO2
or HMF reductones
and free radicals)

+protein-NH2 +protein-NH2 +protein-NH2 +protein-NH2

cross-linked protein aggregates and brown pigments

Colaco CALS et al, ACS Symp Ser, 567 (1994) 222240

 Reducing sugars
71

Glucose
Anomeric carbon atom
CH2OH OH

H O H H CH2OH H
H H
OH H OH H
OH OH O
OH

H OH H OH

Anomeric carbon atom: carbon atom to which two oxygen atoms are covalently linked

When a hydroxyl group is covalently linked to the anomeric carbon atom,

the ring can open by which a reducing group (aldehyde or ketone) is formed
 Reducing sugars
72

Lactose CH2OH

H O OH
H
CH2OH
OH H
Anomeric carbon atom
to which a hydroxyl group
OH O O H
is covalently linked
H Ring can open!
OH H H OH

H H

Anomeric carbon atom

H OH to which NO hydroxyl group
is covalently linked
Ring cannot open!
 Reducing sugars
73

H
CH2OH
O OH
Lactose
H
CH2OH OH H
OH O O H
H
OH H H OH CH2OH
H H H OH H
H
H OH CH2OH OH H
-lactose OH O O O
H
OH H H OH
H H
CH2OH
H OH
H O H
H
CH2OH OH H
OH O O OH
H
OH H H OH
H H

H OH
a-lactose
 Nonreducing sugars
74

Sucrose

Trehalose

No ring opening possible No reducing sugar

 Nonreducing sugars
75

Inulin

No ring opening possible No reducing sugar

 Physico-chemical characteristics
sugars - oligosaccharides
76

Effect molecular weight of inulin on its Tg

180 180 Inulin
140 140 Trehalose

Tg (oC)
Tg (oC)

Sucrose
100 100
Glucose
60 60

20 20
0 2 4 6 8 0.0 0.3 0.6 0.9 1.2
molecular weight (kDa) 1/molecular weight (kDa)

a
Fox-Flory equation: Tg (kDa) Tg,max
Mw(kDa)
Intersection y-axis: maximal Tg possible
Hinrichs WLJ et al, Int J Pharm, 215 (2001) 163-174
 Physico-chemical characteristics
sugars - oligosaccharides
77

Effect molecular weight of inulin on its Tg

-10 -10 Inulin
Trehalose

Tg' (oC)
Tg(oC)

-20 -20
Sucrose

-30 -30 Glucose

-40 -40
0 2 4 6 8 0.0 0.3 0.6 0.9 1.2
molecular weight (kDa) 1/molecular weight (kDa)

a'
Fox-Flory equation: Tg (kDa) Tg,max
' '

Mw(kDa)
Intersection y-axis: maximal Tg possible
 Physico-chemical characteristics
sugars - oligosaccharides
78

Tg and change in mass as a function of the relative

humidity to which the glasses were exposed at 20 oC

80 30

Change in mass (%)

40 20
Tg (0C)

0 10

-40 0
30 40 50 60 0 25 50 75 100
RH (%) RH (%)

Inulin 4 kDa Inulin 1.8 kDa Trehalose

Inulin 2.4 kDa Inulin 1 kDa Sucrose Crystallization !!
(sucrose anhydrate and
trehalose dihydrate)
 Relative humidity
79

The relative humidity of the atmosphere above a

saturated salt solution depends on the type of salt
RH at 25 oC (%) RH at 25 oC (%)
Lithium bromide 6 Sodium bromide 58
Lithium chloride 11 Potassium iodine 69
Potassium acetate 23 Sodium chloride 75
Magnesium chloride 33 Potassium bromide 80
Sodium iodine 38 Potassium chloride 84
Potassium carbonate 43 Potassium nitrate 94
Magnesium nitrate 53 Potassium sulfate 97

Solution: l l RTlnx Specific salt: Specific x

Vapor: v v RTln
p
p Specific p
Equilibrium: v l
Specific RH!
Greenspan L J et al, Phys Chem, 81 (1977) 8996
 Physico-chemical characteristics
oligosaccharides
80

Gordon-Taylor curves of inulin/water mixtures

450
Inulin 4 kDa
400 Inulin 2.4 kDa
Tg (K)

Inulin 1.8 kDa

350 Inulin 1 kDa

300

250
0.85 0.90 0.95 1.00
Weight fraction sugar

wwater . Tg ,water + k . winulin. Tg ,inulin

Gordon-Taylor equation: Tg (mix) =
wwater + k . winulin
 Physico-chemical characteristics
sugars - oligosaccharides
81

Sugar content of the

maximally freeze concentrated fraction
Derived from Gordon-Taylor equation:
Tg,water Tg '
Cg '
Tg ' k 1 k Tg,inulin Tg,water
Glass transition curve
Temperature

Inulin kDa k Cg
Tg
1 0.167 0.84
1.8 0.167 0.82
2.4 0.145 0.84
4 0.186 0.79

Pure water Pure sugar Literature k Cg

Cg
Weight fraction sugar Trehalose 0.13 - 0.19 0.72 - 0.83
Sucrose 0.19 - 0.27 0.65 - 0.83
 General
82

- Physico-chemical properties of oligo- and

polysaccharides for the stabilization of proteins are better

than those of small sugars

- However, small sugars (disaccharidesa) are mostly used

- Reason: most oligo- and polysaccharides are too bulky

and behave like rigid rods

a) As monosaccharides are always reducing sugars

 The Groningen hypothesis
83

- Use oligo/polysaccharides with a

flexible molecular structure

OR
- Use a combination of disaccharides
and oligo/polysaccharides
 The Groningen hypothesis
84

Efficiency and stability sugar coating

Coating by disaccharide
Excellent coating
Poor physical stability

Uncoated
protein Coating by rigid polysaccharide
Poor coating
Excellent physical stability

Coating flexible polysaccharide

Excellent coating
Excellent physical stability

Coating by disaccharide/rigid polysaccharide

Excellent coating
Excellent physical stability
 Flexible molecular
structure of inulin
85

Flexible Rigid
- Backbone outside of sugar ring - Backbone through sugar ring
- Furanose (5) rings - Pyranose (6) rings
 Near infrared spectroscopy
86

Near infrared spectroscopy (NIR) during freeze drying:

- Monitoring apparent water activity (AWA)
- Monitoring H-bonding

If changes in AWA and H-bonding coincide during drying

Excellent water replacement

If changes in AWA and H-bonding do not coincide during drying

Poor water replacement
 H-bonding during
freeze drying by NIR
87
Freeze dry cycle Inulin 4 kDa

Trehalose Dextran 70 kDa

Model protein: LDH

Mensink MA et al, Int J Pharm 496 (2015) 792-800 Black: AWA; grey: Amide A/II frequency
 Miscibility by ssNMR
88

Measuring relaxation time:

- T1 miscibility at 20-50 nm scale
- T1rho miscibility at 2-5 nm scale

T1 T1rho

Yuan XD et al, Mol Pharm 11 (2013) 329-337

 ssNMR freeze dried IgG
89

At 2-5 nm scale protein is only miscible

with trehalose and inulin 1.8 kDa

At 20-50 nm scale protein is only miscible

with trehalose, inulin 1.8 kDa and 4 kDa
and dextran 1.5 kDa

Mensink MA et al, AAPS Journal 18 (2016) 12251232

 Model systems
90

Sugars: Trehalose; disaccharide

Inulin 4 kDa; flexible oligosaccharide
Dextran 6 kDa; rigid oligosaccharide
Dextran 70 kDa; rigid polysaccharide
Dextran 70 kDa/trehalose 1:1 (w/w)

Model proteins: Insulin; 6 kDa

Hepatitis B surface antigen (HBsAG); 25 kDa
Lactate dehydrogenase (LDH); 140 kDa
-Galactosidase (-gal); 540 kDa

Tonnis WF et al, Mol Pharm 12 (2015) 684-694

 Storage stability
91

Stability after storage at 60 oC

- Curve fitting using the equation: f(t) = -A ln (t) + B where f(t) is the activity
relative to the initial activity, t the time in days and A and B are fitting constants.
- A was used as a measure for stability to rank stabilizing capacities of the sugars.
 Curve fitting and ranking
92

Trehalose Dextran Inulin Dextran Dextran No sugar

70 kDa + 4 kDa 70 kDa 6 kDa
trehalose
Ranking stabilizing
capacities sugars
93
 Polysaccharide/disaccharide
mixtures
94

Storage stability of a model protein incorporated in

polysaccharide/disaccharide mixtures by freeze drying

30oC/0%RH 30oC/56%RH

Disaccharide (wt fraction) Disaccharide (wt fraction)

Teekamp N, manuscript in preparation

Thank you for your attention