STABILISING PROTEINS BY SPRAY
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STABILISING PROTEINS BY SPRAY DRYING WITH ADJUVENTS Geoffrey Lee Friedrich 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

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Why spray dry a protein ? The spray drying process: machines & process conditions

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Why spray dry a protein the spray drying process machines process conditions

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 ?


Why spray dry a protein the spray drying process machines process conditions

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.


Why spray dry a protein the spray drying process machines process conditions

The Spray Drying Process


Why spray dry a protein the spray drying process machines process conditions

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


Why spray dry a protein the spray drying process machines process conditions

Laboratory Scale Spray Dryer: Niro Mobile Minor

Drying Capacity up to 7 kg/hr; Maximum Tinlet 350°C; 3' x 6' x 6' high


Why spray dry a protein the spray drying process machines process conditions

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'


Why spray dry a protein the spray drying process machines process conditions

Micro Spray Dryer: Büchi B-290

Drying capacity up to 1.5 kg/h; size 500 x 600 x 1000 mm


Why spray dry a protein the spray drying process machines process conditions

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…


Why spray dry a protein the spray drying process machines process conditions

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 !)


Why spray dry a protein the spray drying process machines process conditions

Example I: SEM Appearance

Residual Moisture = 2.85 % w/w


Why spray dry a protein the spray drying process machines process conditions

Example I: Aggregation status: HPLC of liquid feed


Why spray dry a protein the spray drying process machines process conditions

Example I: Aggregation status: HPLC of Product


Why spray dry a protein the spray drying process machines process conditions

Example I: Secondary structure evaluation with FT-IR


Why spray dry a protein the spray drying process machines process conditions

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


Why spray dry a protein the spray drying process machines process conditions

Example II: SEM appearance

Residual moisture = 4.4/5.0 % w/w


Why spray dry a protein the spray drying process machines process conditions

Example II: Aggregation status: SEC of liquid feed


Why spray dry a protein the spray drying process machines process conditions

Example II: Aggregation status: SEC of product


Why spray dry a protein the spray drying process machines process conditions

Example II: Aggregation status: SEC of formulated product


Why spray dry a protein the spray drying process machines process conditions

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


Why spray dry a protein the spray drying process machines process conditions

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


Why spray dry a protein the spray drying process machines process conditions

Thermal inactivation of catalase


Why spray dry a protein the spray drying process machines process conditions

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


Why spray dry a protein the spray drying process machines process conditions

Stabilizing effects of trehalose on catalase


Why spray dry a protein the spray drying process machines process conditions

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


Why spray dry a protein the spray drying process machines process conditions

Thermal inactivation of lactate dehydrogenase (LDH) in trehalose


Why spray dry a protein the spray drying process machines process conditions

Process & storage stabilities of LDH in trehalose


Why spray dry a protein the spray drying process machines process conditions

Improvement of process stability of LDH in trehalose + 0.1 % g/g Polysorbat 80


Why spray dry a protein the spray drying process machines process conditions

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 ?


Why spray dry a protein the spray drying process machines process conditions

Single droplet drying levitator


Why spray dry a protein the spray drying process machines process conditions

Single droplet drying levitator


Why spray dry a protein the spray drying process machines process conditions

Single-droplet drying kinetics of carbohydate solution

I

II

III

IV


Why spray dry a protein the spray drying process machines process conditions

Single-droplet drying kinetics of maltodextrin (20%)


Why spray dry a protein the spray drying process machines process conditions

Single-droplet drying kinetics of catalase/trehalose


Why spray dry a protein the spray drying process machines process conditions

Final Particles Removed from Levitator

catalase/trehalose (6:4)

catalase


Why spray dry a protein the spray drying process machines process conditions

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 ?


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