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.

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

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

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

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

Case Study: Lyophilized Formulation Development of an

Enzyme

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

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

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)

Formulations Evaluated as part of Study 1

Form. Buffer pH ExcipientsA Tris 7.4 3% Mannitol

1% TrehaloseB Tris 7.4 5% Dextran

C Phosphate 7.4 3% Mannitol1% Trehalose

D Phosphate 7.4 5% Dextran

Lyophilization Cycle – Study 1

-45

-35

-25

-15

-5

5

15

25

35

45

0 5 10 15 20 25 30 35

Time (hours)

Tem

p

60

70

80

90

100

110

120

Va

ccu

m (

mT

orr

s)

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 

Appearance of CakesTr-M/T Ph-M/TTr-D Ph-D

Stability of Enzyme – Post LyoForm Buffer Excipients Osm.

(mOsm/Kg)

NaClAdded (mM)

Moisture (%)

A Tris 3% Mannitol1% Trehalose

217 40 0.2

B Tris 5% Dextran 24 140 <0.1C Phosphate 3% Mannitol

1% Trehalose230 35 0.2

D Phosphate 5% Dextran 30 135 <0.1

Stability of Enzyme – Post Lyo

Sample

Buffer Excipients Assay (%)

A Tris 3% Mannitol1% Trehalose

84

B Tris 5% Dextran 84C Phosphate 3% Mannitol

1% Trehalose143

D Phosphate 5% Dextran 135

Formulations Evaluated as part of Study 2

Sample Buffer pH ExcipientsA Tris 7.4 3% Mannitol

1% TrehaloseC Phosphate 7.4 3% Mannitol

1% TrehaloseE Tris 7.4 4% Trehalose

F Phosphate 7.4 4% Trehalose

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)

Tem

p

50

55

60

65

70

75

80

85

90

95

100

Va

ccu

m (

mT

orr

s)

Shelf In

A

B

C

Cap Man

Pirani (mTorr)

Appearance of Cakes

Tr-M/T Ph-M/T Ph-TrehTr-Treh

Formulations Evaluated as part of Study 2

Form Buffer Excipients NaCl (mM)

Assay 1 (%)

A Tris 3% Mannitol1% Trehalose

40 88

C Phosphate 3% Mannitol1% Trehalose

30 141

E Tris 4% Trehalose 75 98

F Phosphate 4% Trehalose 70 110

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)

Tem

p

50

55

60

65

70

75

80

85

90

Va

ccu

m (

mT

orr

s)

Shelf In

A

B

C

Cap Man

Pirani (mTorr)

Lyophilization with NaCl – Cake Appearance

No impact on appearance when Mannitol is present in crystalline form!

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

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.

ConclusionsInitial Product

Development IssuesImprovements 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