Concentrate on this: Tips for concentration of proteins and peptides

Concentration of proteins and peptides is such an exciting, but challenging application that I have decided to dedicate another post on the topic. Previously, I have discussed different techniques you have available to concentrate proteins. Today, I focus on tips on how to optimize your rotary evaporation or freeze-drying process for concentration of proteins and peptides. Read on and discover these for yourself.

I am in a great mood these days, because Euro 2020 has started! As you might already know from previous blog posts, I am a big soccer and general sports fan. To watch Belgium’s first game, I prepared myself lots of snacks, including microwaveable popcorn. I was setting up my microwave to make the popcorn, when I realized all the different settings for the different types of food you can warm up in there. You have settings optimized for popcorn, pasta, pizza, drinks, defrosting, vegetables and so on. One instrument, many applications.

Funnily, nearly every lab equipment can be equated to a microwave. What do I mean? Well, all can be set up with parameters that are optimized for particular applications. For example, how you use a Rotavapor® for distillation of gin flavours is different from how you set up a Rotavapor® for concentration of proteins. The same is true for your chromatography system, freeze dryer unit and most of your lab equipment.

I just wrote a blog post on concentration of proteins and peptides using freeze drying and rotary evaporation. I think I will then continue with this topic by offering you some tips and tricks on how to set up your rotary evaporator and freeze dryer with parameters best suited for concentration of proteins and peptides.

Tips for using a rotary evaporator for concentration of proteins

  • Optimize the pressure – use a solvent table or a rotary evaporator with an integrated solvent library to look up the recommended values for the solvent you are working with. To avoid bumping and foaming, work slowly up to the desired set value and keep the pressure consistent.
  • Follow the “Delta 20 rule” – I have written about the “delta 20 rule” in rotary evaporation before. To briefly summarize, the rule is used as a guideline to compromise between high evaporation output and energy usage. According to the rule, for concentration of proteins or even other applications, you should set the temperature difference between heating bath and vapor temperature, as well as vapor and cooling, to 20°C. For example, if you are using the chiller at 0°C, you need to set up the vapors at 20°C and the bath at 40°C in order to bring in and carry off the accumulated energy efficiently.
  • Load 75% of your condenser – Optimal condenser loading is 75%, no matter your application and this includes concentration of proteins. At this value, the distillation in balance. In other words, the energy input for evaporation and the energy output through the condenser are in equilibrium. To achieve 75% condenser loading, monitor the height of the condensed solvent on the coils of the condenser.
  • Use a larger flask – A larger flask offers a larger surface area, which positively influences the evaporation performance. Doubling the evaporation flask volume can increase the performance by up to 50% making your concentration of proteins and peptides more effective.
  • Increase rotation speed – Faster rotation substantially increases the surface area available for evaporation, as well as the turbulence in the bath and solvent. More turbulence leads to higher efficiency of heat transfer from the bath.
  • Optimize your process for foaming samples – Proteins tend to foam during rotary evaporation, which could result in product loss. To prevent this from happening during concentration of proteins and peptides, you could use a foam sensor or specialized glass assembly. The foam sensor is an optical sensor mounted on the condenser. The sensor detects foam formation and acts to automatically break the foam by releasing pressure. An “expansion” glass assembly traps any foam that forms into an expansion glass vessel, preventing the foam from reaching the condenser. The expansion vessel must be cleaned after each use.
condenser, rotary evaporation, foaming samples
  • Optimize your process for powder samples – Powder samples, such as those during concentration of proteins, tend to stick to the evaporating flask, leading to product loss. To prevent this problem, you can use drying evaporation flasks. These types of flasks have indents that reduce the accumulation of powder on the glass walls. If you want more information on this topic, check out my previous blog posts on flask sizes and the glass thickness of your flask size and their importance in rotary evaporation.
rotary evaporation, laboratory evaporation, lab evaporation, drying flask

In addition, if available, you could use the Drying mode on the regulating interface. The Drying mode alternates rotation direction in user-defined intervals for a defined period of time to support drying a slurry and increasing the efficiency during concentration of proteins. For more information on this topic, check out my previous blog post on how to set up the interface.

Tips on using a freeze dryer for concentration of proteins

  • Keep the distance between the freezer and freeze dryer short – For concentration of proteins, place the freezer and freeze dryer in the same room to reduce the time of sample transfer and maintain the frozen sample at a low temperature. If possible, perform a leak test on your freeze dryer to detect misconfigurations and defects.
  • Increase product surface area – In general, the drying rate of a product depends on the surface area relative to the volume of product. The larger the surface area, the faster the product dries, since more water molecule can leave the matrix. If you want to increase product surface when using manifold freeze-drying for concentration of proteins, I recommend using shell freezing so that the product is spread on the inner wall of the flask while freezing. Experimental data shows sublimation rates are two times faster when shell freezing is used instead of bulk freezing. Read my previous blog post on using shell freezing to speed up manifold freeze drying for more information.
  • Decrease fill depth – The container surface and fill depth affect the lyophilization process. In freeze drying, a sublimation front moves from the top of the sample (air/solid interface) towards the bottom of the sample vial, resulting in a porous powder. The speed at which the sublimation front moves depends on the depths of the sample. During the freeze-drying process for concentration of proteins, the amount of dried sample progressively grows larger on top of the frozen sample. However, a larger dried sample cake increasingly hinders the vapor from escaping from below. The thicker and larger the cake, the more of an impediment it presents. This causes several problems including, slowing down of the process, affecting sample temperature or premature melting of the sample before it is dried. To alleviate these effects and improve your concentration of proteins, I recommend increasing the surface and decreasing the sample depths.
  • Choose the right freezing temperature – The majority of products undergoing freeze drying consist of a solvent, such as water, and some material, the solute, dissolved or suspended in water. The freezing temperature of such a formulation, typical for concentration of proteins, is determined by its characteristics and composition. When cooling the mixture, water is the first to separate from the substances as it freezes to ice. The formulation may then appear frozen, but the remaining substance could still be liquid. These substances form concentrated areas that eventually freeze at temperatures below the freezing point of water. The temperature where all components of the mixture are properly frozen is the maximum temperature the formulation can endure the lyophilization. Importantly, if you apply vacuum to an incompletely frozen eutectic mixture, you might cause destruction of the product as unfrozen components expand when placed under vacuum.

I hope you have managed to stay concentrated during all these tips and tricks. I know it is a lot of information, but I do hope you find it useful in optimizing your concentration of proteins and peptides. As for myself, I think I will be popping out of here now to enjoy some more popcorn in front of a soccer game. See you soon science and soccer fans!

Till next time,

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