Are you team “spray dry” or “freeze dry” when you formulate proteins and peptides?

I have decided it’s finally time to formulate a post on how to formulate proteins and peptides. To do this, I give you a quick overview of two favourite techniques for dehydrating proteins: spray drying and freeze drying. And I stay focused with direct comparisons of the methods and real-life examples of protein formulations using spray drying and freeze drying.

It’s no secret I’m a bit of a sports fan. I’ve written about watching the World Cup and Euro 2020 and arguing about hockey . Now that I am in retirement, I can enjoy even more sports. In fact, to my greatest enjoyment, I recently watched an exciting Formula 1 race with some friends.

But I can’t help it that besides liking sports, I also like science. And somehow the name Formula 1 made me think about chemical formulas! And then about formulations. And then I realized it is about time I finish my protein/peptide series with you by doing a post on how to formulate proteins and peptides.

We did already discuss how to purify proteins and how to concentrate proteins. The next and final step in the process would be how to formulate proteins:

protein; peptide; concentration; purification; formulation; spray drying; chromatography; freeze drying; evaporation; rotary evaporation

One of the most common reasons we need to formulate proteins and peptides is for pharmaceutical applications. In drug formulations, several substances are combined with an active ingredient. This active ingredient, however, may not be directly incorporated into a formulation, as it may be unstable or display unwanted qualities. One way to facilitate storage before incorporation in a final formulation is to pre-formulate the ingredient either in a liquid or in a solid form.

The type of technique you chose to dry a biopharmaceutical usually depends on the final use of the compound, compound characteristics, financial aspects and production capacity and requirements.

The most common techniques used to formulate proteins and peptides are spray drying and freeze drying. Let us first compare these with a general overview:

 Spray dryingFreeze drying
Drying timeShort - secondsLong - days/weeks
Yield50 - 80% on lab scale units100% in vial
Product temperature50⁰C or above>0⁰C - primary drying
20 - 40⁰C - secondary drying
StressesShear, atomization, air-liquid, interfacial, mechanical, thermal, dehydrationIce crystal formation (solid-liquid interfacial stress), change of ionic strength and pH, phase separation, dehydration, crystallization
Control of particle characteristicsYesNo
Capital costModerateVery high
Operating costModerateHigh
ProductionContinuousBatch

And let us explore each technique in a bit more detail.

Using spray drying to formulate proteins

Spray drying is the preferred solution for drying many small molecules and for amorphous solid dispersions. The advantages of the technique, including low cost and particle engineering possibilities, make it an attractive choice for the drying formulations of biopharmaceuticals. The method is high-throughput, continuous, easy to scale and cost-efficient.

In terms of finances, investment and operational costs of spray drying when you formulate proteins are about 4 to 7 times lower than those of freeze drying, even though hot gas is generated, and the process consumes a high amount of energy.

As I just said, the ability to control the particle characteristics of the dried powder by changing process parameters make the technique ideally suited for applications such as pulmonary and nasal delivery, where particle characteristics are of particular importance. Using spray drying to formulate proteins enables a good processability of the powder into tablets or capsules after being mixed with excipients.

How does spray drying work when you formulate proteins?

Spray drying is accomplished by dissolving, emulsifying or dispersing a core substance in a solvent or in a solution of carrier material. The material is then atomized and sprayed into a a drying chamber, where a stream of hot drying gas evaporates to the solvent to produce dry solid particles. These solid particles are further separated from the gas stream and collected.

Since the transformation of a liquid product into a dry powder is achieved in a single step, spray drying is advantageous whenever you formulate proteins, because of costs, scale-up and process simplification. You can readily generate tablets or capsules without milling or downstream processing steps. Since spray drying exposes biomolecules to shear, air-liquid, interfacial and dehydration stresses, most temperature-sensitives, including enzymes, proteins and antibiotics, can be spray dried without suffering major activity loss.

One disadvantage of using spray drying to formulate proteins is yields in laboratory scale. Since you suffer product loss on the wall of the drying chamber and into the exhaust air, yields are typically in the range of 20 to 70%.

Use of high-efficiency cycles can increase yields to reach over 80%.

However, bear in mind that insufficient forces of liquid atomization and the inability of the cyclone to effectively separate fine particles with a diameter below 2 μm, makes production and recovery of sub-micron particles challenging. Laboratory-scale spray drying is also unsuitable for production of particles with a size range above 50 μm, similar to those produced at large scale.

Using freeze drying to formulate proteins

Freeze drying, also known as lyophilization, is an effective way of drying material. The method is based on the physical principle of sublimation, which involves the direct transition between solid and the gaseous phase, bypassing the liquid phase. The frozen sample is dried under vacuum, without any thawing in-between. Freeze drying is particularly suitable for applications such as the preservation of delicate material against degradation or decomposition, the preservation of product characteristics and original shape, the conservation of products that require fast rehydration or the conditioning of a product for further use.

The key parameters to control when you formulate proteins using freeze drying include pressure and temperature. How you set these parameters is governed by sample characteristics.

A typical freeze-drying process consists of two stages, freezing and primary drying. In some cases a third stage, called secondary drying, might be required to remove solvent molecules tightly attached to the sample and to reduce moisture content further.

freeze drying; lyophilization

Most of the water is removed from the sample by the end of the primary drying phase.

Regardless if you formulate proteins or other samples, the residual moisture content of the product is typically around 5-10% due to water bound to the matrix.

After the primary drying phase, ice should be not be present in the sample anymore. The secondary drying step removes the adsorbed water molecules by desorption. To achieve ideal conditions for desorption, the lowest possible pressure and high shelf temperatures are required. When you select the shelf temperature, you should also consider effects on product stability. The secondary drying stage is usually faster than the primary drying stage.

At the end of secondary drying when you formulate proteins, the product moisture content should be in the range of 1-5%.

In terms of pharmaceutical applications, freeze drying is usually the preferred method for the preservation of a wide range of drug compounds, especially when stability in the liquid state is inadequate and whenever the formulations do not require further processing, as they are filled directly in vials, which are sealed in the drying after the cycle to prevent potential contamination.

Freeze-drying operates at low process temperatures and leads to high yields, great product uniformity, high quality in terms of activity, water content and stability. You obtain a product with very high quality, since freeze drying reduces the risks of intrinsic product properties, such as collapse, eutectic melt or glass transition temperatures being exceeded.

At this point, I’d like to point out how you can use the two techniques if you want to formulate proteins. Here a few examples of real-life protein formulations prepared by spray drying and freeze drying:

ProcessProteins/peptidesStabilizers
Freeze dryingIgGTrehalose, Sucrose, PEG
Freeze dryingLyzozymePEG, Glycerol, Sucrose, Trehalose, Dextran
Freeze dryingBSAGlucose, Sucrose, Maltose, Trehalose, Maltotriose
Freeze dryingAnti-IgE antibodyHistidine, Arginine, Glycine, Aspartic acid
Spray DryingIgGTrehalose, Sucrose, Leucine, Glycine, Lysine, Phenylalanine
Spray DryingTrastuzumabTrehalose, HPβCD, βCD
Spray DryingAnti-IgE Mab, rhDNaseMannitol, Trehalose, Sucrose
Spray DryingCatalaseArginine, Histidine, Glycine
Spray DryingInfluenza vaccineHEPES buffer, Phosphate buffer
Spray DryingAlkaline phosphataseSodium carboxy methylcellulose
Spray DryingEPODextran

Now if you would rather de-focus and look at the whole techniques as a whole, I recommend you glance over the Spray Drying: Basics & Applications free guide and the Freeze Drying Guide: Volume 1 and Volume 2. You are also very lucky, my colleagues will be having a webinar on the topic of purification, concentration and formulation of proteins and peptides in November, so you are free to watch this resources as well.

And with this, I think it’s time to slam on the breaks, as I am blabbing quite a bit here. If you haven’t yet decided which technique is right for you, have no fear. I have not exhausted all I have to say on this topic. Tune in, I promise to follow-up with another champion of a post on tips and tricks on how to formulate proteins and peptides using spray drying and freeze drying. These should certainly help you make a more informed choice in finding the ideal technique for applications where you formulate proteins.

Till next time,

The Signature of Bart Denoulet at Bart's Blog