Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
STABILISING PROTEINS BY SPRAYDRYING WITH ADJUVENTS
Geoffrey Lee
Friedrich Alexander University, Erlangen
1. Why spray dry a protein ?2. The spray drying process: machines & process conditions3. Two examples of spray-dried pharmaceutical proteins4. Sources of damage to proteins during spray drying5. Formulation measures to stabilize proteins
carbohydrates, surfactants6. Single droplet drying via levitation7. Is spray drying a potentially useful process for my product ?
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
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.
Why Use Spray Drying for Proteins ?
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
The Spray Drying Process
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
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
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Laboratory Scale Spray Dryer: Niro Mobile Minor
Drying Capacity up to 7 kg/hr; Maximum Tinlet 350°C; 3' x 6' x 6' high
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
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'
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Micro Spray Dryer: Büchi B-290
Drying capacity up to 1.5 kg/h; size 500 x 600 x 1000 mm
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
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…
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
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 !)
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Example I: SEM Appearance
Residual Moisture = 2.85 % w/w
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Example I: Aggregation status: HPLC of liquid feed
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Example I: Aggregation status: HPLC of Product
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Example I: Secondary structure evaluation with FT-IR
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
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
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Example II: SEM appearance
Residual moisture = 4.4/5.0 % w/w
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Example II: Aggregation status: SEC of liquid feed
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Example II: Aggregation status: SEC of product
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Example II: Aggregation status: SEC of formulated product
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Potential sources of protein damage
Liquid feed
Nozzle Atomizing air
Drying tower
1. Adsorption
2. Shearing forces
3. Liquid/air interface expansion
4. Thermal stress
Drying air
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
The 2 periods of droplet drying
Constant-rate phaseT = approx. Twetbulb
Critical point
Falling-rate phaseT Toutlet
Various morpholgies
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
eg, Tinlet/Toutlet = 130oC/90oCResidence time: 1s – 25s
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Thermal inactivation of catalase
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
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
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Stabilizing effects of trehalose on catalase
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
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
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Thermal inactivation of lactate dehydrogenase (LDH) in trehalose
Tinlet/outlet [oC] 90/60 110/70 130/80 150/95
Aktivity t = 0 89 3 (n=4) 92 6 (n=4) 82 9 (n=4) 76 8 (n=4)
w [% g/g] 4.7 3.9 2.9 2.4
Tg [oC] 65 77 78 85
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
-4 0 4 8 12 16 20 24 28 320
102030405060708090
100110120130
97/23
4 °C 40 °C 25 °C 60 °C
Resi
dual a
ctivi
ty [%
]
Time [weeks]
Process & storage stabilities of LDH in trehalose
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
0 4 8 12 16 20 24 280
102030405060708090
100110120130
97/24
4 °C 40 °C 25 °C 60 °C
Resi
dual a
ctivi
ty [%
]
Time [weeks]
Improvement of process stability of LDH in trehalose + 0.1 % g/g Polysorbat 80
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Development of a spray drying process for a protein
1. 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 ?
2. 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 ?
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Single droplet drying levitator
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Single droplet drying levitator
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Single-droplet drying kinetics of carbohydate solution
I II
III IV
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Single-droplet drying kinetics of maltodextrin (20%)
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Single-droplet drying kinetics of catalase/trehalose
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
Final Particles Removed from Levitator
catalase catalase/trehalose (6:4)
Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin
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 conditions
has a (fairly) sound scientific basis.5. The product capacity can be adjusted within wide boundaries.6. The powder properties can also be taylored by process
or 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 ?