1.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin STABILISING PROTEINS BY SPRAY DRYING WITH ADJUVENTS Geoffrey Lee Friedrich 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 ?

2.  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 ?

3.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin The Spray Drying Process

4.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Process Variables Control Product Quality Independent process variables: - Drying Air Inlet Temperature, T inlet - Drying Air Relative Humidity, % RH - Drying Air Flow Rate, v da - Liquid Feed Flow Rate, V lf - Atomising Air Flow rate, v aa Dependent variable: - Outlet Air Temperature, T outlet

5.  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 T inlet 350°C; 3' x 6' x 6' high

6.  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'

7.  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

8.  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 T in /T out = 110 o C/70 o C - ca 20% loss of monomer (SEC)  2 further examples of pharmaceutical proteins illustrate use of analytical techniques…

9.  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 !)

10.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Example I: SEM Appearance Residual Moisture = 2.85 % w/w

11.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Example I: Aggregation status: HPLC of liquid feed

12.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Example I: Aggregation status: HPLC of Product

13.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Example I: Secondary structure evaluation with FT-IR

14.  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

15.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Example II: SEM appearance Residual moisture = 4.4/5.0 % w/w

16.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Example II: Aggregation status: SEC of liquid feed

17.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Example II: Aggregation status: SEC of product

18.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Example II: Aggregation status: SEC of formulated product

19.  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

20.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin The 2 periods of droplet drying Constant-rate phase T = approx. T wetbulb Critical point Falling-rate phase T  T outlet Various morpholgies Spray dryer typeDroplet diameter [µm]  constant-rate [s]  falling-rate [s] Micro-Laboratory (Büchi)  10 µm 0.0010.002 Pilot machine100 µm0.040.1 eg, T inlet /T outlet = 130 o C/90 o C Residence time: 1s – 25s

21.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Thermal inactivation of catalase

22.  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, T g  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

23.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Stabilizing effects of trehalose on catalase

24.  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

25.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Thermal inactivation of lactate dehydrogenase (LDH) in trehalose T inlet/outlet [ o C]90/60110/70130/80150/95 Aktivity t = 0 89  3 (n=4)92  6 (n=4)82  9 (n=4)76  8 (n=4) w [% g/g]4.73.92.92.4 T g [ o C]65777885

26.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Process & storage stabilities of LDH in trehalose

27.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Improvement of process stability of LDH in trehalose + 0.1 % g/g Polysorbat 80

28.  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 ?

29.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Single droplet drying levitator

30.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Single droplet drying levitator

31.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Single-droplet drying kinetics of carbohydate solution I II III IV

32.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Single-droplet drying kinetics of maltodextrin (20%)

33.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Single-droplet drying kinetics of catalase/trehalose

34.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Final Particles Removed from Levitator catalase catalase/trehalose (6:4)

35.  Lee, Stabilising Proteins by Spray Drying with Adjuvents, 'Particle 2006' Berlin Summary & Conclusions 1.Spray drying is one of a number of processes that can be used for the production of fine particles. 2.It is an established technique where much expertise and experience 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 ?