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 ?