Stabilization of Protein Drugs During

Lyophilization Process –
Theoretical Concepts and Practical Considerations

Madhav Kamat Ph.D., R.Ph

Kamat Pharmatech LLC

6th International Conference on Lyophilization and Freeze Drying,

Brazil, Oct 2013
 Degradation Pathways of Proteins

• Physical
– Denaturation or unfolding
– Adsorption
– Aggregation
• Chemical
– Aggregation (co-valent bonded)
– Deamidation
– Isomerization
– Disulfide exchange
– Oxidation
– Fragmentation
– others
 More Common Degradation Pathways of
Proteins
• Physical
– Denaturation or unfolding
– Aggregation
– Adsorption
• Chemical
– Aggregation (co-valent bonded)
– Deamidation
– Isomerization
– Disulfide exchange
– Oxidation
– Fragmentation
– others
 Aggregation
• Complex formation that leads to loss of activity and/or
immunogenicity
• Mechanism:
– Aggregation takes place through hydrophobicity, hydrogen
bonds, and sometimes covalent bonds
• Factors:
– Temp, pH, ionic strength, conc.
• Effect:
– Soluble and/or insoluble aggregates (particulates)
– Super-potency
– Immunogenicity
 Deamidation
• Removal of ammonia from Asn (more common) and Gln to form
respective carboxylic acids/esters
• Mechanism:
– In acidic pH: direct hydrolysis
– In neutral to alkaline pH: formation of intermediate cyclic imide and
eventual isomerization
• Factors:
– Protein structure (accessible Asn), neighboring residues, Temp,
alkaline pH
• Effects: May vary
– Some proteins may show reduced activity by almost 25X
– Some may show immunogenicity
• Analysis:
• IEF, IEX, Tryptic Peptide Mapping (TPM)
 Oxidation
• One of the major pathways for protein degradation
• Methionine to its sulfoxide
• Sulfhydryl to disulfide
• Mechanism:
– Not very well understood
• Factors:
– Light, Trace metals, pH, temperature, buffer
• Effect:
– May result in reduced activity: e.g. fully oxidized hGH
shows only only one fourth activity
• Analysis:
– TPM
 Stability Consideration during Development of
Lyophilized Protein Formulation

• Solution for lyophilization:

– Bulk drug storage
– manufacturing hold time
– contact surfaces
• During Lyophilization:
– Freezing
– Dehydration
• Solid state stability during storage
 Mechanism of Stabilization in Lyophilized Products

• Immobilizing protein as well as other reactants as solid by

removal of water will retard most chemical reactions
• Control of headspace with inert gas
• Use of proper excipients: buffers, antioxidants etc.
• Fundamentally Different Mechanisms:
– During freezing/frozen state
– During drying phase
– During Shelf life storage
 Stabilizers of Proteins in Solutions
• Stabilizers in solution tend to stabilize the native structure

• Excellent early works by

– Arakava, Timasheff, Carpenter etc.

• Mechanism:
• Differential hydration or exclusion of the solvent
• Stabilizers exhibit negative isotherms on the protein
surface and are excluded. To reduce the chemical
potential, protein tends to minimize surface and
thereby prefer native state.
 Stabilizers of Proteins in Solutions

• Result of maintaining native state:

– Compact (native) structure: related to Tm’ (transition

temperature mid-point) as measured by DSC
– Reduces exposure of hydrophobic surfaces
– Hides reactive functional groups
– Accessibility for reaction sites is is reduced

• For other bulk reactions: Buffers, Ionic strength, Anti-oxidants

 DSC: Transition Temperature Mid-point (Tm’).
Model mAb

Ref buffer: Tris, NaCl, EDTA, pH 7.4 Low pH buffer: Citrate, pH 3.0 After adj from pH 3.0 to 6.0 with Tris
 Stabilizers of Proteins during Freeze-drying

• Facts:
– Stabilizers in solution tend to stabilize during freezing/frozen state and
vice-a-versa
– Stabilizers during freezing/frozen (cryo-protectants) state may not act
as stabilizers during dehydration phase (lyo-protectants) and vice-a-
versa
– Some compounds do act as both Cryo- and lyo-protectants

• Hypotheses:
– Differential hydration or exclusion of the solvent
– Vitrification (glass or amorphous) matrix
– Water replacement/Hydrogen bonding
 Changes During Freezing/Frozen State
• Ice phase
– Ice formation/separation (exothermic change)
– Development of large ice-aqueous interface area
• Interstitial Phase:
– concentrates leading to solutes either precipitate as crystalline or amorphous
phases, or becomes supersaturated phase, or turns into a glass
– pH change (phosphate buffer – dibasic ppt leads to lower pH)
– Large changes in ionic strength possible
– Redistribution of solutes: depends on rate and direction
– Large increase in viscosity of the unfrozen phase. However, molecular mobility
does remain for proteins
• Protein Stress
– Surface induced damages
– concentration effects
– cold denaturation
• Result:
– Conformational changes to proteins
– Protein aggregation (soluble and insoluble), precipitation, visible particulates,
Changes During Freezing of 3% Sucrose solution

Bhatnagar 2007
14
 pH Shifts during freezing

Precipitation of
Dibasic salt

Monobasic salt

Bhatnagar 2007
15
Stabilizers of Proteins during Freezing/Frozen Stage
During Freezing/Frozen state
• Vitrification or amorphous phase is necessary
• Protein must reside in the amorphous phase
• Dilution of the protein in a rigid, inert glassy solid
• Dilution separates protein molecules from each other
• High viscosity matrix reduces molecular mobility
• Dilution also minimizes the interstitial fluid stress – pH effects,
ionic strength, et.
• Stabilization is a kinetic mechanism
 Changes During Drying/Storage
• Ice sublimes during primary drying
• Non-ice is removed during secondary drying
• Protein surface (bound) moisture is removed and H-bonds are
disrupted
• Tg gradually starts moving up as the moisture reduces

Result:
– Conformational changes to proteins
– Protein aggregation (soluble and insoluble), precipitation,
visible particulates,
 A. Crystalline Trehalose
B. Lysozyme + Trehalose lyophilized
C. BSA + Trehalose lyophilized
D. Trehalose hydrated

A. Crystalline Trehalose, B. FD Lysozyme + Trehalose, C. FD BSA + Trehalose, D. Hydrated Trehalose

Proteins during FD behave as water behaves with Trehalose

Arakawa et al: 2001
Amide band regions for hydrated lysozyme, FD lysozyme alone, and FD lysozyme with trehalose

Arakawa 2001
Chang and Pikal 2009
 Stabilizers of Proteins during Dehydration
During dehydration phase
• Water replacement theory
• Replacement of hydrogen bonding because of the vacating
water molecules is necessary
• Glass formation in the dried state
• Destruction of hydration shell results in irreversible loss of
activity of many labile proteins
• Many cryo-protectants fail to protect during dehydration
Development of a stable freeze-dried formulation of recombinant human interleukin-1
Receptor antagonist by Chang et al (1996)
Development of a stable freeze-dried formulation of recombinant human interleukin-1
Receptor antagonist by Chang et al (1996)
 Cryoprotectants for proteins

Sugars Sucrose, lactose, glucose, trehalose, maltose

Polyols Inositol, ethylene glycol, glycerol, sorbitol, xylitol, mannitol
Amino acids Sodium glutamate, proline, alanine, glycine, lysine
Polymers Polyethylene glycol, dextran, polyvinylpyrrolidine
Inorganic salts Sodium sulfate, ammonium sulfate, potassium phosphate,
magnesium sulfate
Organic salts Sodium acetate, sodium polyethylene, sodium caprylate,
propionate, lactate, succinate
Miscellaneous Trimethylamine N-oxide, sarcosine, betaine, GABA,
strombine, DMSO, ethanol

Ref: T. Arakava et al. 2001

 Lyo-protectants for proteins

Sugars Sucrose, lactose, glucose, trehalose, maltose

Polyols Inositol, ethylene glycol, glycerol, sorbitol, xylitol, mannitol
Amino acids Sodium glutamate, proline, alanine, glycine, lysine
Polymers Polyethylene glycol, dextran, polyvinylpyrrolidine

Ref: T. Arakava et al. 2001

 Development Process: Typical lyophilized product

Drug Stability Pharmaceutical Support

Property (Use-time and Mfg time)
Enhancement techniques Biological Manufacturing/
Considerations Handling

None
(for Hydrolysis Stable)
. Lyo Process Development
. Tonicity
. Physicochemical Properties Yes . Compatibility Studies
. Hemolysis . Diluents, IV-sets,
. Mode and Speed of Action Stability In-line filters
No . Precipitation . Manufacturing Surfaces
. Dose requirement
. Phlebitis . Use-time stability
. Buffer
. Pain on injection . Photo-stability
. Bulking Agent

. Cryo-/lyo-protectants

. Anti-oxidant

. Chelating agent

. Co-solvent

(effect on Tc and Tg’)

Modified from Chapter, “Formulation of small and large volume parenterals” by Kamat and DeLuca
 Solution Formulation Development Studies

• Perform formula optimization studies

• Perform solution compatibility studies with containers, in-line

use filters, diluents, and administration sets

• Generate stability data:

– Real-time, stress, accelerated and Use time

• Identify critical quality attributes and preliminary process

parameters.
 Product Development Studies
• Product/Container Compatibility
• Sterilizing filters compatibility
• Container Integrity Test
• Process/Equipment Machinability Studies
• Cleaning/Sanitization/Fogging Agents Interference Studies
• Processing Times: API and Excipients mixing times, formulation time,
filtration time
• Bulk Solution Holding Times
• Sterile Holding Time
• Head Space Studies
• Process/Equipment Specificity Studies
• Definition of preliminary operational parameters
• Product/manufacturing contact material Compatibility Studies
• Photo-Stability Studies
• Storage/Shipping Studies
Formulation Development of Lyophilized product

Solution product development

AND

Lyophilization process development

and Cycle design

Both activities are inter-dependent

 Formulation Excipient Selection

Formulation excipients must:

– Stabilize the drug
• Cryo-protection and/or Lyo-protection
– Be a Justified Additive: Tonicity, Buffer, Preservative, etc.
– Favorably affect critical temperatures
– Improve cake structure: bulking agent
– Aid in mass transfer
– Be chemically inert
– Acceptable for injection use
– Low endotoxin and low bioburden quality

Must be consistent with the lyophilization cycle objectives

Practical Considerations in Development of FD protein products

• Accurate determination of critical thermal transitions (e.g.,

Teu, Tg’, Tc)

• Annealing usually helps in drying rate:

– Improves frozen structure to help mass transfer,
releases water (forms ice)

• Assess effect on product quality if critical temperature is

exceeded (if only a minor portion is “microcollapse”, doe it
matter to the overall quality)

• Effect of the fill height:

– Lower the better
– Larger diameter vials preferred: will reduce throughput
Practical Considerations in Development of FD protein products

• Effect of the cake density:

– Less concentrated solution preferred
– Too low concentration will result in fluffy cake and some
product may escape out of vial
– If low concentration:
• Use “higher” chamber pressure
• Tight control over solenoid /metering bleeding valve
• Use thoroughly dried stoppers

• Use of bulking agent: Mannitol, Lactose, Sucrose, Glycine,

Dextran
– Provides structure/channels
– Modifies Tc
– Must consider movement of Tg with respect to water content
– Sucrose and Trehalose are most common
Practical Considerations in Development of FD protein products
• Protein concentration: Denaturation at ice-water interface is “limited”.
More protein, less percentage will be denatured

• Buffer choice: Avoid sodium phosphate or potassium phosphate

• Tonicity: Mannitol, Glycine better than NaCl (due to better Tg and other
effects)

• Increasing protein amounts generally increases Tg’ (Tg’ of pure protein

solutions is about -10 ºC), however, other components are needed in the
formulations to stabilize it and the Tg is lowered.

• Cooling rate:
– Faster cooling leads small ice crystals (reduced mass transfer across
dry layer), greater ice surface area (more instability due to cold-
denaturation)

– Slower cooling rates may result in larger ice crystals (better mass
transfer across dry layer), re-distribution of solute, formation of mass-
transfer-impeding skin
Practical Considerations in Development of FD protein products

• Use stabilizer that has a high Tg (during storage) and has

less dependence on water
• Trehalose is more resistant to hydrolysis than Sucrose
• Use of surfactant to prevent aggregation throughout the
manufacture (filling, filtration)
• Beware of crystallization of mannitol during transient
short period high temperature excursion leading to
release of water for hydrolysis
• Use of surfactant to prevent hydrophobic interactions –
silicone issues with marketed products (Orencia, Nulojix)
• Use of vacuum to prevent foam
Practical Considerations in Development of FD protein products

Pay special attention to:

• Temperature ramp from primary drying phase to

secondary drying

• Many experimental runs are needed to arrive at

synchrony between the raising of shelf temperature and
loss of residual moisture and its impact on the cake
structure

• Movement of Tg in this region is critical

Practical Considerations in Development of FD protein products

• Once the formulation and process parameters are set:

minor tweaks to the cycle are done by keeping the
following TRIAD of parameters in mind:
– Appearance
– Residual Moisture
– Reconstitution time
• Once these are fixed, generally the quality of the product
(physico-chemical) is assured.
Examples of some lyophilized products in the market
 Remicade

• Infliximab Active 100 mg

• Sucrose Cryoprotectant 500 mg
• Polysorbate-80 Surfactant 0.5 mg
• Monobasic sodium Buffer 2.2 mg
phosphate, monohydrate
• Dibasic sodium
phosphate, dihydrate Buffer 6.1 mg
 Earlier Experience with Infliximab

Laboratory experiments in 1991

 Orencia

• Abatacept Active 250 mg

• Maltose Cryoprotectant 500 mg
• Polysorbate-80 Surfactant 0.5 mg
• Monobasic sodium Buffer 17.2 mg
phosphate, monohydrate
• Sodium Chloride Bulking agent 14.6 mg
 Herceptin IV

• Trastuzumab Active 440 mg

• Trehalose Stabilizer 400 mg
• L-Histidine Stabilizer 9.9 mg
• L-Histidine.HCL Stabilizer 6.4 mg
• Polysorbate-20 Surfactant 1.8 mg
Thank You Very Much!
Please contact me if you have any questions at:

madhav.kamat@kamatpharma.com

Or

www.kamatpharma.com
 Solution Stability and stabilization

Conformational Stability of Lysozyme: Effect of NaCl

Sodium Chloride Transition Calorimetric Specific
Temperature, Tm Enthalpy, Enzyme
(oC) DH (Cal/mol) × 104 Activity
(EU/mg) × 103
Control 77.1 ± 0.1 9.6 ± 0.4 47.0 ± 1.5
0.05M NaCl 77.0 ± 0.1 9.0 ± 0.3 45.0 ± 1.0
0.06M NaCl 76.7 ± 0.2 8.9 ± 0.5 44.2 ± 1.0
0.12M NaCl 76.5 ± 0.2 8.8 ± 0.5 41.0 ± 1.7
0.17M NaCl 76.0 ± 0.6 8.4 ± 0.1 40.9 ± 0.9
0.50M NaCl 75.1 ± 0.5 7.5 ± 0.9 37.0 ± 1.0
0.83M NaCl 73.7 ± 0.6 5.9 ± 0.5 29.0 ± 1.8
Solution Stability and stabilization
Solution Stability and stabilization
Solution Stability and stabilization
Solution Stability and stabilization
Development of a stable freeze-dried formulation of recombinant human interleukin-1
Receptor antagonist by Chang et al (1996)
Development of a stable freeze-dried formulation of recombinant human interleukin-1
Receptor antagonist by Chang et al (1996)
Development of a stable freeze-dried formulation of recombinant human interleukin-1
Receptor antagonist by Chang et al (1996)