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STABILISING PROTEINS BY SPRAYDRYING WITH
ADJUVENTSGeoffrey LeeFriedrich Alexander
University, Erlangen

• Why spray dry a protein ?
• The spray drying process machines process
  conditions
• Two examples of spray-dried pharmaceutical
  proteins
• Sources of damage to proteins during spray drying
• Formulation measures to stabilize proteins
  carbohydrates, surfactants
• Single droplet drying via levitation
• Is spray drying a potentially useful process for
  my product ?

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Why Use Spray Drying for Proteins ?

• Spray drying (SD) of protein-containing systems
  is not new !
• Advantages robust, standard equipment, process
  development straightforward, relatively
  inexpensive, scale up Disadvantages needs
  exact in-process control, yield optimization
  required, minimization of deposit formation,
  'continuous' process.
• Applications of SD proteins in pharmacy -
  inhaleable powders - injectable powders -
  stable, flowable storage-form for bulk protein.

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The Spray Drying Process
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Process Variables Control Product Quality
Independent process variables -
Drying Air Inlet Temperature, Tinlet
- Drying Air Relative Humidity, RH
- Drying Air Flow Rate, vda - Liquid
Feed Flow Rate, Vlf - Atomising Air
Flow rate, vaa Dependent variable -
Outlet Air Temperature, Toutlet
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Laboratory Scale Spray Dryer Niro Mobile Minor
Drying Capacity up to 7 kg/hr Maximum Tinlet
350C 3' x 6' x 6' high
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Pilot Scale Spray Dryer Niro P6.3
Drying capacity up to 60 kg/hr Chamber 1.6 m x
0.8 m x 60 Size 11.5' x 9' x 15'
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Micro Spray Dryer Büchi B-290
Drying capacity up to 1.5 kg/h size 500 x 600 x
1000 mm
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Feasibility of spray drying a protein

• Product quality (peptide/protein) investigated
  by - activity loss (enzymes) - change in
  aggregation status (HPLC, SEC) - gel
  electrophoresis eg, isoelectric focussing -
  alteration in FT-IR amide bands
• Example model protein trypsinogen (Tzannis
  Prestrelski, 1999) - ca 15 activity loss on SD
  at Tin/Tout 110oC/70oC - ca 20 loss of
  monomer (SEC)

• 2 further examples of pharmaceutical proteins
  illustrate
• use of analytical techniques

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Example I A low molecular weight peptide

• Substance Peptide with 20 amino acids ca. 2.5
  kDa
• SD conditions - Büchi 191 Micro Spray
  Dryer
• Liquid Feed - 2 mg/mL peptide (very low !)

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Example I SEM Appearance
Residual Moisture 2.85 w/w
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Example I Aggregation status HPLC of liquid feed
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Example I Aggregation status HPLC of Product
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Example I Secondary structure evaluation with
FT-IR
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Example II A high molecular weight protein

• Substance IgG (AMG 162) with MWt ca. 150 kDa
• SD conditions - Büchi 191 Micro Spray
  Dryer
• Liquid Feed - 115 mg/mL IgG ? Sorbitol

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Example II SEM appearance
Residual moisture 4.4/5.0 w/w
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Example II Aggregation status SEC of liquid feed
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Example II Aggregation status SEC of product
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Example II Aggregation status SEC of formulated
product
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Potential sources of protein damage
Drying air
2. Shearing forces
Nozzle
Atomizing air
1. Adsorption
Liquid feed
3. Liquid/air interface expansion
4. Thermal stress
Drying tower
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The 2 periods of droplet drying
Various morpholgies
Critical point
Constant-rate phaseT approx. Twetbulb
Falling-rate phase T ? Toutlet
Residence time 1s 25s
eg, Tinlet/Toutlet 130oC/90oC
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Thermal inactivation of catalase
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Primary formulation measure to reduce protein
damage

• Glass-forming carbohydrates or amino acids ? can
  reduce level of protein damage -
  prevent unfolding aggregation - which
  carbohydrate/protein mass ratio ? ? during SD
  water replacement mechanism ? after SD glassy
  immobilisation ?
• Low residual moisture content ? ensures high
  glass transition temperature, Tg ? important for
  protein storage stability
• Sufficient storage stability of carrier ? ?
  amorphous systems are hygroscopic ? must
  prevent moisture uptake crystallisation ? also
  deterioration in powder properties

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Stabilizing effects of trehalose on catalase
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Secondary formulation measures

• Addition of surfactant to liquid feed ? can
  reduce protein surface excess at
  water/air interface of atomised liquid feed
• Use of non-aqueous solvents ? for peptides with
  low aqueous solubility ? higher w/v improves
  particle formation
• Polymers to taylor particle morphology ? eg,
  dextrans or hydroxy ethyl starches ? eg,
  surfactants

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Thermal inactivation of lactate dehydrogenase
(LDH) in trehalose

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Process storage stabilities of LDH in trehalose
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Improvement of process stability of LDH in
trehalose 0.1 g/g Polysorbat 80
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Development of a spray drying process for a
protein

• Laboratory scale SD of protein solution -
  sufficient water solubility alternative
  solvent - does damage to protein occur ?
  (aggregation) - residual moisture content
  OK ? - which process conditions give best
  result ?
• Which formulation measures are necessary ?
  - do I need a carbohydrate ? Probably yes.
  - which protein/carbohydrate weight ratio ?
  (maximize) - adjustment of required
  particle size useage ?
• 2. Move to pilot scale machine - depends
  on required process throughput (kg of
  powder per h) - upscale increases
  residence time in chamber - can I use the
  same nozzle setup ?

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Single droplet drying levitator
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Single droplet drying levitator
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Single-droplet drying kinetics of carbohydate
solution
I
II
III
IV
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Single-droplet drying kinetics of maltodextrin
(20)
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Single-droplet drying kinetics of
catalase/trehalose
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Final Particles Removed from Levitator
catalase/trehalose (64)
catalase
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Summary Conclusions

• Spray drying is one of a number of processes that
  canbe used for the production of fine particles.
• It is an established technique where much
  expertise andexperience is available.
• Development can be performed under GMP
  conditions.
• The selection of a suitable machine process
  conditionshas a (fairly) sound scientific basis.
• The product capacity can be adjusted within wide
  boundaries.
• The powder properties can also be taylored by
  processor formulation.
• Potential problems some questions need to be
  addressed - how do I obtain a high product
  yield ? - how do I minimize protein damage
  ? - how much stabilizing adjuvent do I need,
  and which one is the best for my protein
  ? - what is the patent situation ?