Principles and Applications of
Lyophilization to Biotechnology
Michael J. Akers, Ph.D.
Baxter BioPharma Solutions
Bloomington, Indiana
BIOMAN 2010 Conference
Ivy Tech Community College
Bloomington, Indiana
July 13, 2010
1
Baxter Confidential
Organization of Presentation
Baxter Briefs
Biologics
Market
Lyophilization
2
Baxter Confidential
Bloomington, Indiana Capabilities Overview
Technologies
Prefilled syringes
Lyophilization
Aseptic liquid vials
Cartridges
Diluent syringes
Services
 Formulation development
 Process development
 Lyo cycle development
 Analytical development
 Regulatory support
 Labeling and Packaging
Clinical to Commercial
3
Baxter Confidential
Other Interesting Facts about
Baxter BioPharma in Bloomington
• Produce more prefilled syringes than any
other manufacturer in North America
• Small molecules, vaccines, proteins,
diluents
• Produce injectables marketed throughout
the world
• No history of FDA Warning Letters
• 308 employees in 2001; 993 end of 2009
• Newest manufacturing building won ISPE
facility of the year in 2006
Focus of Bloomington R&D
 Formulation Development of Small Volume Injectables
 Liquid
 Lyophilized
 Extensive experience with
 Proteins
 Monoclonal Antibodies
 Small Molecules
Approximately 80%
of projects in past 2 y
 Analytical Method Development
 Method development and validation services available
 Responsible for validation of transfer of all incoming QC
methods for Bloomington facility
 Expertise in cleaning validation limit calculations and method
development/validation
 Development stability studies
 Lyophilization Process Optimization Research Programs
5
Baxter Confidential
Bloomington R&D Known As Baxter
Lyophilization Center of Excellence
DSC Thermogram of EC90
Endotherm Up
20 W/g
2 W/g
Heat Flow, W/g
We characterize APIs by thermal analyses and
freeze-dry microscopy to develop formulations
and lyophilization cycles
-30
We use freeze-dry microscopy and FTIR to
evaluate formulation effects on protein stability
-60
-55
-50
-45
-28
-26
-40
-35
-24
-22
-30
-20
-25
-18
-20
-16
-15
-10
-5
0
5
10
15
20
25
Temperature, °C
We coordinate transfer and scale up of labdeveloped product to production freeze-dry
operations.
Our research programs include mechanisms of
heat and mass transfer during freeze-drying,
nucleation optimization, process monitoring by
Tunable Diode Laser Absorption Spectroscopy
Since 2001, 35 peer-review research articles, 3
books, 5 book chapters, dozens of short courses,
and 80 presentations at conferences and
universities
Front Door
1
2
Chamber
Test
Section
Detectors
Condenser
~ - 80°C
to
Vacuum
Vacuum
Pump
H-3236
6
Baxter Confidential
Baxter BioPharma Solutions
Pharmaceutical Research and Development
Tim Paul, Lisa Hardwick, Nathan Pease, Wendy Saffell-Clemmer, Larry Callahan
Bloomington, IN Top:
Middle: Rhonda Haley,Wei Kuu, Melissa Beard, Mike Hildreth, Karen Abram, Kevin O’Bryan, Elizabeth Oslos, Michael Akers
Bottom: Lindsay Stanford, Angie Kruszynski, Nathaniel Fair, Jackie Karty, Greg Sacha, Hoang Mitchell, Dan Staples, Kelly Roby, Steven Nail
7
Baxter Confidential
Market segment by therapeutic area
and molecule class
45,000
Market Size of Biological
40,000
35,000
mAb Sales 2010 ($m)
30,000
Protein Sales 2010 ($m)
25,000
Sales ($M)
Total
20,000
15,000
10,000
5,000
0
ab
Di
gy IID logy ase ogy NS ular alth es tor y inal her
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A o se tol C sc he cin ra st Ot
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n
m
a o
e ec
as
C
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W
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f
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s
ete
Source: Baxter Analysis
Baxter Confidential
8
Current/Future Facts About Biologics
• Significant growth
 2005 sales: $33B
 2005 predicted 2010 sales: $82B
 1 out of 4 new drugs introduced in US/EU is a biopharm
product
• Monoclonal antibodies (MoAbs)
 $30B of 2010 sales expected to be MoAbs
 > 200 MoAbs in clinical development right now
• About half of current and new biologic products require
lyophilization
• Many of these products will be self-administered
• Obviously, there is a huge market potential for simple, costeffective devices to be used in home health care to prepare and
deliver lyophilized biologics (and other lyo products)
 Especially if product is for chronic health care
9
Baxter Confidential
Best Known (Best Selling)
Lyophilized Biologics (Biopharmaceuticals)
Product Brand (Indication) Company
2009 U.S. Sales
Enbrel (Arthritis)
Amgen
$6,580,000,000
Remicade (Arthritis, Crohn’s)
J&J
$5.934,000,000
Herceptin (Breast cancer)
Genentech
$4.890,000,000
7 Human Growth Hormone
Brands (Growth hormone
Pfizer, Novo, Lilly,
Genentech, Serono,
Sandoz, Ipsen
$2,886,000,000
Avonex (Multiple sclerosis)
Biogen Idec
$2,323,000,000
Advate (Hemophilia)
Baxter
$2,058,000,000
Betaferon (Muliple sclerosis)
Berlex/Bayer/Merck
$1,649,000,000
Total Biologic Sales
---
deficiency)
$91,780,000,000
10
Baxter Confidential
Lyophilization
• The terms “lyophilization” and “freeze-drying” used
interchangeably
• Started in the 1930s, importance grew in WWII
• Lyophilization is the conversion of a liquid to a solid
through the process of sublimation
• A sterile solution is prepared, filled into primary
containers, placed into a freeze-dryer, and frozen
• The solvent in the solution is removed by directly
converting it from frozen ice to water vapor.
• What remains in the container are the solute
components in the solid state containing very low
residual moisture (typically 0.5-2.0%).
11
Baxter Confidential
Schematic Overview of Processing
Solution and Freeze-Dried
Biopharmaceutical Dosage Forms
Dispense raw materials
(active and excipients)
Prepare solution in appropriate
Thaw and pool
mixing tank (add ingredients to
Water For Injection)
Wash and sterilize
active biopharmaceutical
primary containers
and closures
Add active to solution,
ISO 8, Grade D,
pH adjustment, final QS
Class 100,000
This is formulation bulk solution
ISO 5, Grade A
Sterile filter formulated
Class 100
bulk solution
Aseptically fill formulated bulk solution
Transfer to Freeze-dryers
into primary package and stopper
and lyophilize
(partial stopper if product is to be freeze-dried
Fully insert stopper, remove
from freeze dryer
Apply aluminum overseal
Storage at appropriate
100% inspection
temperature (usually 2-8ºC)
Baxter Confidential
Label, sec package,
storage, distribution
12
Preparing Product for Lyophilization









Prepare the sterile solution—compound,
mix, filter
Fill into containers, typically vials
Partially insert a special designed rubber
closure onto the vials
Aseptically load the vials into a freeze dry
chamber
Freeze every single solution in every vial
below a pre-determine critical temperature
Using appropriate application of
temperature and pressure, sublime the ice
from the product
Using further application of temperature and
pressure, remove the necessary amount of
bound water from the product
Automatically stopper the vials, neutralize
the chamber
Aseptically remove the vials from the
chamber and apply aluminum seals
Baxter Confidential
13
Today’s State of the Art Technology Uses
Automated Systems To Transfer Product
From Production Line Into the Freeze-Dryer
14
Freeze-Dryer Subsystems (What Must Be
Operating Smoothly and Maintained for
Lyophilization Process To Succeed?)
•
•
•
•
•
•
•
Heat Transfer System
Refrigeration System
Vacuum System
Stopper-in-Place System
Clean-in-Place System
Control System
Loading/Unloading System
Typical Batch of a Lyophilized Biopharmaceutical Product
Worth Several Million $
Baxter Pharmaceutical Solutions
15
Baxter Confidential
Courtesy of Samir U. Sane, Ph.D., Genentech, Inc.
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Baxter Confidential
Product temperature lower than
shelf temperature during primary
drying because energy is consumed
as ice sublimes to vapor.
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Baxter Confidential
Flow of Vapor from Chamber to Condenser
T
T
P
Vacuum
Pump
Water
Vapor
Flow
Door
Product
Chamber
Condenser
Chamber
Connecting Duct
Condenser Coils
Mushroom Valve
(open position)
Shelves with
Vials
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Baxter Confidential
Schematic of Heat and Mass Transfer
in the Freeze Dryer
Temperature difference between chamber and condenser and pressure differential
Heat Transfer
Conversion of solid
(ice) to vapor in
chamber called
sublimation
Mass Transfer
between solution in vials and vacuum pump drives ice out of vial and onto the condenser
Vacuum Pump
ΔP
Dry Cake
Sublimation Front
Frozen Solution
Thermal Fluid
Condenser
Pressure gradient between
sublimation front and chamber
Shelf
Thermal fluid circulates within the shelves to
control temperature in chamber
Baxter Confidential
19
Desired Freeze Dried Product Characteristics
Intact cake
Sufficient strength
Uniform color
Sufficiently dry
Sufficiently porous
Sterile
Free of pyrogens
Free of particulates
Chemically stable—both in dry
state and after reconstitution
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Baxter Confidential
Advantages of Lyophilization





Removal of water at low
temperature
Ease of reconstitution
Compatible with aseptic operations
More precise fill weight control
Done properly, the freeze-dried
solid has relatively high specific
surface area, which promotes rapid,
complete reconstitution
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Baxter Confidential
Disadvantages of Lyophilization
• The drug may not be stable as a
freeze-dried solid
• Many biological molecules are
damaged by the stresses
associated with freezing, freezedrying, or both
• Not all solutes can be freeze-dried
to form a pharmaceutically
acceptable cake
• Cost may be an issue, depending
on the product
22
Baxter Confidential
Common Lyophilized Products
• Pharmaceuticals – large and small
molecules
• Bacteria
• Viruses
• Vaccines
• Plasma
• Small Zoological Specimens
(Taxidermy)
• Fruit
• Coffee
• Flowers
• Water-Damaged Documents
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Baxter Confidential
Main Challenges of Lyophilization
• Development of formulation that meets all the necessary
critical product attribute requirements (quality appearance,
potency, stability, recon time, etc)
• Accurate determination of the “critical temperature” of final
formulation necessary to determine conditions of
lyophilization cycle (Tg’, Te, Tc)
• Establishment of temperature, pressure, and time cycle
settings that can achieve best product quality in shortest
possible time
• Transfer and scale-up of lab-developed process to
production scale process
• Keep all the equipment running smoothly
• Special challenges in product development of lyophilized
biologic formulations and cycles
24
Challenges in Lyophilization of Protein
Pharmaceuticals
Baxter Pharmaceutical Solutions
25
Baxter Confidential
Challenges in Lyophilization of Protein
Pharmaceuticals
• Measurable differences in
recovery of activity can be
associated with differences in
the thermal history of freezing
• Some proteins can be subject to
overdrying; that is dryer is not
necessarily better
• Stability of the freeze dried solid
is often a concern. Most freeze
dried proteins require
refrigerated storage.
• Solid state stability can be
affected by small differences in
residual moisture content.
Baxter Confidential
26
Why Does Lyophilization Take So Long?
• Sometimes, the properties of the
formulation require that the
temperature be very low, often
below –30oC. This decreases the
driving force
• The heat required is very high, and
heat transfer in freeze drying is very
inefficient
• Resistance to mass transfer –
transport of water vapor from the
sublimation front through the porous
bed of partially dried solids – can be
significant.
• Huge batch sizes, takes time to
complete the 3 stages of lyophilization
Process Data for Representative Freeze-Dry Cycle
30
300
20
250
10
200
-10
-20
150
-30
Thermocouples
100
-40
-50
50
-60
-70
0
0
10
20
30
40
Time (hours)
50
60
70
Pressure (mTorr)
Temperature (°C)
0
Final Comments
• Roughly 50% of all commercial biologic (therapeutic
protein products) are lyophilized.
• Lyophilization technology and the expertise to use it
are vital to the ability of the biopharmaceutical
industry to prepare and market life-saving injectable
medicines
• Lyophilization is the most challenging (and most
expensive) of all sterile product manufacturing unit
operations
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Baxter Confidential
Acknowledgements
• Thanks to all my Baxter R&D colleagues for
the great collegiality we have and what
makes my job so enjoyable.
• Thanks to the following individuals for
providing some of the slides I showed
 Dr. Steven Nail
 Ms. Lisa Hardwick
 Ms. Wendy Saffell-Clemmer
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Baxter Confidential