The Importance of Understanding the Physical State of Excipients in a FreezeDried Formulation: Implications for Overall, Product Quality Juan Davagnino, Ph.D. Biopharmaceutical Development, KBI Biopharma, Inc.

1. The Importance of Understanding the
Physical State of Excipients in a Freeze-
Dried Formulation: Implications for Overall
Product Quality
Juan Davagnino, Ph.D.
Biopharmaceutical Development
KBI Biopharma, Inc.

2. Lyophilized Formulation Development
• Necessary for products susceptible to storage-induced
physical and chemical degradation
• Removal of water reduces mobility and eliminates
water as a reactant
• Typically necessary to include the following:
• Buffer (minimal)
• Bulking agent
• Stabilizer
• API
• Should minimize total solids (<10% w/v) content in
order to minimize resistance to mass transfer during
drying

3. Common Lyo Components and
Physical State
• Crystalline
• Bulking agent – mannitol, glycine
• Salt – NaCl, buffer salt in high enough concentrations
• PEG
• Amorphous
• Saccharides – sucrose, trehalose, lactose, etc.
• Polymers – dextran, PVP, etc.
• API – protein, peptide
• Plasticizers (i.e. sorbitol) and Tg modifiers (i.e. HES)
• Buffer salts – diluted into amorphous phase remain
amorphous

4. The Lyo Process – Impact on Phase
Behavior
• Low temperatures
• Nucleation and growth of ice results in concentration
of solute
• Annealing during freezing may allow for phase
separation in concentrated state
• Annealing allows for crystallization of crystalline
bulking agents, i.e. mannitol, glycine
• Crystallization of excipients can occur during freezing
or early in primary drying
• Crystallization of salts can occur – potential for pH
shift

5. Potential issues
• Amorphous phase separation may result in separation
of protein and intended stabilizer
• Crystallization of a component intended to remain
amorphous may result in stability and aesthetic
concerns, i.e. cake collapse
• Improper balance of amorphous and crystalline
components may result in delayed or absence of
crystallization
• High amorphous salt concentrations will suppress the
Tg´ making the product more susceptible to collapse
and increase drying times

6. Case Study: Lyophilized
Formulation Development of an
Enzyme

7. Formulation Issues on Phase I Product
• Lyophilization cycle needs optimization
• The lyophilized product had to be stored at
-15°C to maintain stability
• Poor lyophilized product appearance
• pH instability at low ionic strength

8. Client Requirements
• Lyophilized formulation
• Low concentration of enzyme
• Same activity as current formulation (1X PBS)
• High NaCl upon administration
• Need lowest attainable osmolality in lyo product to allow for
recon in NaCl diluent
• Need to study the addition of NaCl in lyo product
• Aesthetically elegant cake

9. Standard Approach
• Evaluate various formulation buffers, pH, and
excipients
• Narrow down buffer and excipient selections
• Determine ratio of excipients or total excipient
concentration ideal for lyo:
• Ratio of crystalline: amorphous should be at least 3:1
• Total solids should not be less than 3% (w/v)

10. Formulations Evaluated as part of Study 1
Form. Buffer pH Excipients
A Tris 7.4 3% Mannitol
1% Trehalose
B Tris 7.4 5% Dextran
C Phosphate 7.4 3% Mannitol
1% Trehalose
D Phosphate 7.4 5% Dextran

11. Lyophilization Cycle – Study 1
-45
-35
-25
-15
-5
5
15
25
35
45
0 5 10 15 20 25 30 35
Time (hours)
Temp
60
70
80
90
100
110
120
Vaccum(mTorrs)
Shelf In
A
B
C
D
Cap Man
Pirani (mTorr)
Sample
ID Buffer pH Excipient(s)
A Tris 7.4 3%(w/v) Mannitol + 1% (w/v) Trehalose
B Tris 7.4 5% (w/v) Dextran
C Phosphate 7.4 3%(w/v) Mannitol + 1% (w/v) Trehalose
D Phosphate 7.4 5% (w/v) Dextran

12. Appearance of Cakes
Tr-M/T Ph-M/TTr-D Ph-D

13. Stability of Enzyme – Post Lyo
Form Buffer Excipients Osm.
(mOsm
/Kg)
NaCl
Added
(mM)
Moisture
(%)
A Tris 3% Mannitol
1% Trehalose
217 40 0.2
B Tris 5% Dextran 24 140 <0.1
C Phosphate 3% Mannitol
1% Trehalose
230 35 0.2
D Phosphate 5% Dextran 30 135 <0.1

14. Stability of Enzyme – Post Lyo
Sam
ple
Buffer Excipients Assay (%)
A Tris 3% Mannitol
1% Trehalose
84
B Tris 5% Dextran 84
C Phosphate 3% Mannitol
1% Trehalose
143
D Phosphate 5% Dextran 135

15. Formulations Evaluated as part of Study 2
Sample Buffer pH Excipients
A Tris 7.4 3% Mannitol
1% Trehalose
C Phosphate 7.4 3% Mannitol
1% Trehalose
E Tris 7.4 4% Trehalose
F Phosphate 7.4 4% Trehalose

16. Lyophilization Cycle – Study 2
-45
-35
-25
-15
-5
5
15
25
35
45
0 5 10 15 20 25 30 35 40 45 50
Time (hours)
Temp
50
55
60
65
70
75
80
85
90
95
100
Vaccum(mTorrs)
Shelf In
A
B
C
Cap Man
Pirani (mTorr)

17. Appearance of Cakes
Tr-M/T Ph-M/T Ph-TrehTr-Treh

18. Formulations Evaluated as part of Study 2
Form Buffer Excipients NaCl
(mM)
Assay 1
(%)
A Tris 3% Mannitol
1% Trehalose
40 88
C Phosphate 3% Mannitol
1% Trehalose
30 141
E Tris 4% Trehalose 75 98
F Phosphate 4% Trehalose 70 110

19. Lyophilization with NaCl – Cycle time
-55
-45
-35
-25
-15
-5
5
15
25
35
45
0 10 20 30 40 50 60 70 80 90 100
Time (hours)
Temp
50
55
60
65
70
75
80
85
90
Vaccum(mTorrs)
Shelf In
A
B
C
Cap Man
Pirani (mTorr)

20. Lyophilization with NaCl – Cake
Appearance
No impact on appearance when Mannitol is present in
crystalline form!

21. Addition of NaCl to Trehalose formulation results in
contraction/collapse!

22. Summary of Lyo Studies
• The Lyo cycle in the
presence of NaCl is too long
and difficult to transfer to a
manufacturing lyophilizer
• The final formulation
contains 10 mM Phosphate,
4% Trehalose pH 7.4 and it
is reconstituted with 70 mM
NaCl to be isotonic.

23. Conclusions
Initial Product
Development Issues
Improvements Achieved
Lyophilization cycle
needed optimization
The lyophilization cycle was
optimized for the proposed
formulation
The lyophilized product
had to be stored at -15°C
to maintain stability
The product has good stability
at 2-8°C
Poor lyophilized product
appearance
Elegant, homogenous product
appearance
pH instability at low ionic
strength
Stable at isotonic ionic strength