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STABILISING PROTEINS BY SPRAYDRYING WITH
ADJUVENTSGeoffrey LeeFriedrich Alexander
University, Erlangen

1. Why spray dry a protein ?
2. The spray drying process machines process
   conditions
3. Two examples of spray-dried pharmaceutical
   proteins
4. Sources of damage to proteins during spray drying
5. Formulation measures to stabilize proteins
   carbohydrates, surfactants
6. Single droplet drying via levitation
7. 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

1. 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
2. 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

1. Substance Peptide with 20 amino acids ca. 2.5
   kDa
2. SD conditions - Büchi 191 Micro Spray
   Dryer
3. 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

1. Substance IgG (AMG 162) with MWt ca. 150 kDa
2. SD conditions - Büchi 191 Micro Spray
   Dryer
3. 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
Spray dryer type Droplet diameter µm ?constant-rate s ?falling-rate s
Micro-Laboratory (Büchi) ? 10 µm 0.001 0.002
Pilot machine 100 µm 0.04 0.1
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

1. 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 ?
2. Low residual moisture content ? ensures high
   glass transition temperature, Tg ? important for
   protein storage stability
3. 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

1. Addition of surfactant to liquid feed ? can
   reduce protein surface excess at
   water/air interface of atomised liquid feed
2. Use of non-aqueous solvents ? for peptides with
   low aqueous solubility ? higher w/v improves
   particle formation
3. 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
Tinlet/outlet oC 90/60 110/70 130/80 150/95
Aktivity t 0 89 ?3 (n4) 92 ? 6 (n4) 82 ? 9 (n4) 76 ?8 (n4)
w g/g 4.7 3.9 2.9 2.4
Tg oC 65 77 78 85

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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

1. Spray drying is one of a number of processes that
   canbe used for the production of fine particles.
2. It is an established technique where much
   expertise andexperience is available.
3. Development can be performed under GMP
   conditions.
4. The selection of a suitable machine process
   conditionshas a (fairly) sound scientific basis.
5. The product capacity can be adjusted within wide
   boundaries.
6. The powder properties can also be taylored by
   processor formulation.
7. 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 ?