Dr.

Margaret Doherty
Biopharmaceutical Manufacturing
Process
 • Formulation = preparation of the final drug product

• Formulation of the purified bulk drug substance (BDS)

serves several purposes:
• making it possible to administer the active
Formulation pharmaceutical ingredient into humans

• increase the shelf life

• making the final drug product fit to the device

used for administration
Formulation

• Drug products are typically formulated as:

• liquid solutions (e.g. insulin pen systems),
• lyophilized products (human growth
hormone)
• suspensions (several vaccines)

• The final formulation depends on the stability

of the drug product, which is generally
enhanced by lyophilization or immobilization
(e.g. alhydrogels)
• Most dominant destabilizing factors are
aggregation, oxidation, de-amidation and loss
of tertiary structure resulting in decreased
potency
• Excipients can be added in order to increase
protein stability upon freezing, lyophilization
and storage
 The goal of the formulations scientist is to identify

the right formulation conditions, the right

excipients in optimal quantities, and the right

Formulation dosage form to maximize stability, biological

activity, safety, and marketability of a particular

product
The Purpose of Formulation
• The term “formulation, fill and finish”
refers to the series of processing steps that
are needed to turn the purified bulk drug
substance into the final dosage form, the
finished product, for the market.

• The formulation step involves taking the

purified protein and dispensing it with the
correct excipients that can ensure product
quality and integrity during the subsequent
fill/finish steps.
Formulation

• A successful formulation
strategy builds on solid
knowledge on drug
administration, delivery
devices, dose, intended shelf
life, single versus multi-dose
and storage conditions

• The starting point for any

formulation is the drug
substance defined as the
active pharmaceutical
ingredient (API) bulk resulting
from the purification process
 • The first step is to thaw the bulk drug substance (BDS)

• Then it is to formulate it with the right buffer and to the

target concentration

• This involves adding the desired excipients at target

Formulation concentration and adjusting pH and protein

Step concentration

• Sometimes it is operationally more favourable to store

a drug substance at a higher concentration than the
drug product, and therefore a dilution step would be
needed during formulation
 • In other cases, a buffer exchange may be
required between drug substance and drug
product

• Similarly, a concentration step may also be

Formulation needed if the drug product is formulated at a
Step concentration higher than that of drug
substance

• A UF/DF step may then be required

 Pharmaceutical excipients are
substances other than the
pharmacologically active drug which
are included in the manufacturing
process i.e. Excipients are the inactive
materials of pharmaceutical dosage
forms.

Excipients
The behaviour of the dosage form
depends on the interrelationship
among the various excipients in the
formulation and on their collective
effect on the active ingredients.
 Improving palatability or appearance of the active
ingredient

Increasing the stability of active ingredients

The Function
Of Excipients In Acting as a filler/bulking agent so that an adequate
A amount of material is available to properly fill a dosage
Pharmaceutical form
Dosage Form Acting as an antimicrobial or antioxidant to extend the
shelf life

Enabling/enhancing delivery of a drug or medicine and

thus improving its efficacy, bioavailability, uniformity,
and/or flow characteristics
Assisting the manufacturing process e.g. improving flow
Excipients used in parenteral dosage forms and their
function
How do we deal with these issues?
 Low temperature reduces
microbial growth and metabolism

Low temperature reduces

denaturation

Storage - Low temperature reduces

Refrigeration adsorption

Freezing is best for long-term

storage

Freeze/Thaw can denature proteins

 • Most protein formulations deliver either:

• Liquid

Protein • Frozen liquid

Storage • Buffered aqueous protein solutions
may be stable ~ 2 yrs

• Solid Lyophilized (freeze-dried) protein

Protein Storage

Proteins in
solution often do
not meet the The abundant A biologically
preferred stability presence of water active protein
requirements (>2 promotes suitable for long-
years), even chemical and term storage is
when kept physical often by
permanently degradation lyophilisation
under refrigerator processes (freeze-drying)
conditions (cold
chain)
Lyophilisation

• Lyophilization is the process of

removing water from a product

• Reduces chemical or biological

degradation

• Extends the shelf life of

biopharmaceuticals

• Leaving a final water content <

3% in powder form
 Main Considerations:

• Stress test determination – is lyophilisation

actually needed

• It is an expensive process - last resort

Why do we need
• Key process driver: product stability
Lyophilisation?
• How will product be delivered – vial or syringe
system

• Many biotech products stable only if lyophilised

or frozen
 • long-term stability

• short reconstitution time

Desired • elegant cake appearance

characteristics • maintenance of the characteristics of
of a the original dosage form upon
lyophilized reconstitution, including:
product
– solution properties

– structure or conformation of
proteins

– particle-size distribution of
suspensions
Principles of Lyophilisation

• The fundamental principle in freeze-

drying is sublimation, the shift from a

solid directly into a gas

• Sublimation of water takes place at

temperatures & pressures below the

triple point

Sublimation vs. Melting

Triple Point

The triple point of a

substance is the point

were 3 phases liquid,

solid and vapour

coexist at particular

temperature and

pressure
 • There are 4 stages in the complete drying process:

• Pre-treatment
• Freezing
• Primary drying
• Secondary drying

Freeze drying process

1. Pre-treatment

• Includes any method of treating prior to freezing

• Solute phase concentration

• Formulation to preserve product appearance

• Formulation to increase surface area etc...

2. Freezing

• The purpose of the freezing stage is to convert most of the water

in the liquid formulation into ice and all solutes (protein,
excipients, etc) to solids

• The product is frozen by lowering the shelf temperature, and

hence lowering the temperature of the primary packaging in
contact with the shelf (typically a glass vial)
 2. Freezing
• As ice crystals form the solutes are
compressed/concentrated in the areas
between these ice crystals until they
crystalize (some excipients) or become an
amorphous solid (proteins and some
excipients).

• The temperature at which this occurs is

known as the Eutectic temperature (Te), for
crystalline solutes, or glass transition
temperature (Tg), for amorphous solutes.
2. Freezing
• Once the Te or Tg has been met and
the formulation is solid, primary drying
can begin

• Typically, a temperature of –40˚C is

reached prior to reducing the pressure
in the chamber to approximately
50μbar (absolute/vacuum) before the
primary drying/sublimation begins
 2. Freezing
Rate of cooling important
• Rate of freezing has a direct impact on the
size of the ice crystals formed

• Fast or supercooling forms small ice

crystals
• Slower cooling rates form larger ice
crystals ~ possibly results in damage to
the fragile protein product

• Prevent precipitation of excipients

• Prevent pH or ionic shifts
• Prevent ‘cold’ denaturation of the protein
product
• Primary drying is the part of the freeze-drying cycle in which
unbound solution is removed through sublimation

▪ After freezing, the product is placed under vacuum and gradually

heated which allows the ice to evaporate without melting the
product. This process, is called sublimation and transforms the ice
directly into water vapor, without first passing through the liquid
state.

3. Primary Drying (Sublimation)

 The water vapor given off by the product is collected on a condenser,
with a (substantially) lower temperature than the shelf with the vials.

3. Primary Drying (Sublimation)

 Sublimation phase
(Primary drying)

• Temperature drops are avoided by the

supply of heat from the shelf to the vial, so
the shelf is heated during this stage.

• Heat is transferred to the vial through:

1. direct shelf vial contact
(conductance)
2. radiation heat transfer from the
shelf to vial wall
3. gas conduction (varies with
pressure) form the air space
between the shelf and the vial
base.
 ▪ A final but important point for primary drying is
the product temperature is never allowed to
exceed its eutectic or glass transition
temperature range. Typically, a safety margin of
2–5 °C is used
Primary
Drying ▪ If this temperature is exceeded, ice in the vial
(Sublimation) will begin to liquefy, a condition known as melt
back will occur

▪ The liquefied solvent will form a skin over the

product and trap moisture within it

▪ The end result will be an unstable product

 • When all frozen or amorphous water that is
non-protein and non-excipient bound is
removed, the secondary drying step starts

Primary • The end of primary drying is determined

Drying when the product temperature approaches
(Sublimation) that of the shelf temperature

• By the end of primary drying, most products

have lost 95% of their moisture

• Necessary to remove as much the remaining

moisture as possible – by desorption
• In the secondary drying stage, the temperature is slowly increased to remove
‘bound’ water; the chamber pressure is still reduced.

• Typically, the shelf temperature is raised 20/30 °C above the final primary
drying temperature. (Because all the loose water has been removed, There is
no longer a danger of melt back)

• The temperature should continually stay below the collapse/eutectic

temperature

4. Secondary Drying
Secondary Drying
▪ Ends when the product has been kept at 20 °C.
The residual water content is a critical endpoint
indicating parameter. Values as low as 1%
residual water in the cake have been
recommended.

▪ After freeze drying, samples of the product are

taken and tested to ensure that the product is
the correct strength and volume, and it is sterile
(free from microorganisms).
Recap -Four stages in the lyophilisation process
of protein formulations

Freezing The temperature of

the product is reduced from
Pretreatment Includes any ambient temperature to a
method of treating prior to temperature below the triple
freezing point. This ensures
sublimation rather than
melting.
Primary drying Crystallized &
Second drying Removal of
water not bound to
water interacting with the
protein/excipient is removed
protein and excipients. The
by sublimation. The
temperature in the chamber is
temperature is below the Te or
kept below Tg and rises
Tg; the temperature is for
gradually, e.g., from −40 °C to
example −40 °C and reduced
20 °C.
pressures are used.
 • Lyophilisation can stress proteins

• Protein unfolding may occur

Lyophilisation
Process • Stabilising additives are

Concerns required e.g.

Lyoprotectants – glucose,
lactose, sucrose
 • For formulations where a highly potent protein
is used in a low dose (<1%), bulking agents are
required to provide strength to the porous cake
and to prevent blowout

• Blowout - the cake collapses due to movement

Lyophilisation of water vapour in the porous cake, and may
& Excipients cause removal of a portion of the protein
product out in the vapour

• Bulking agent excipients added to the

formulation, e.g mannitol and glycine
 • Excipients which prevent the formed cake from
collapsing at the temperatures typically used
during drying - dextran and gelatine

Lyophilisation • Lyoprotectants are added to the formulation to

& Excipients protect the protein structure from cold

denaturation and degradation resulting from
change to an amorphous state e.g. sugars &
albumin
Lyophilisation Excipients
Successful
Lyophilisation
 Lyophilisation
Advantages

Advantages Disadvantages
Does not denature High capital cost
proteins
Lyophilization is the only
Stability of powder High energy costs
way to deliver stable,
form
biologically active
Rapid and easy Long process time
products with long shelf- reconstitution
life Need for a sterile
diluent upon
reconstitution
 1. Melt-back
2. Maintaining Sterility
3. Maintaining Fill volume
4. Stoppering the product
5. Leakage
Problems 6. Wear and tear on Machinery

commonly
• Melt-back is when some freeze-dried product in
experienced
the vial returns to the liquid state. This is caused
with
by:
Lyophilisation
• Increase in heat content
• Alteration in the vapour escape rate
• Loss of vacuum prior to completion of primary
drying
• Increased instability, poor solubility, increased
reconstitution time
Summary of Formulation and Filling
Process
Addition of
Excipients
1. Adding excipients
2. Sterilising the product (0.2um Sterile
filter) Filtration and
Aseptic Filing
3. Freeze-drying the product if
required. Products may be
formulated into liquid form but
are less stable Liquid
Lyophilisation
Product
4. Sealing of the final product
container, labelling and
packaging Sealing of
Labelling and
final Product
5. The result of final formulation is a Packaging
biopharmaceutical product ready Container
to be used by the patient