Biostabilization

Thermodynamics and Kinetics of Freezing

We use infrared and Raman spectroscopy to determine the kinetic and thermodynamic phenomena in the freeze-concentrated liquid during freezing, vitrification, and thawing. This gives us unique access to molecular level changes proteins, and enzymes experience when they interact with their environments at low temperatures and crowding. Of specific interest is their interections with the ice interface. Some of our recent papers on this topic are:

 

mians paper

FTIR Analysis of Molecular Changes Associated with Warming Injury in Cryopreserved Leukocytes

[Langmuir, 2018, DOI: 10.1021/acs.langmuir.8b02982]

dmso image

 

In Situ Spectroscopic Quantification of Protein–Ice Interactions

[J Phys Chem B, 2013, 117 (26), 7889]

 

dmso image

 

Effect of high DMSO concentration on albumin during freezing and vitrification

[RSC Adv, 2017, 7, 43611]

mannitol

 

Effects of Excipient Interactions on the State of the Freeze-Concentrate and Protein Stability

[Pharm Res, 2017, 34, 462]

DSC

 

Mutual Influence of Mannitol and Trehalose on Crystallization Behavior in Frozen Solutions

[Pharm Res, 2016,  33, 1413]

Room Temperature Storage of Archival Serum Samples

Many cancer biomarkers in the bood get damaged during long term storage when kept at cryogenic temperatures due to stresses associated with ice formation and low temperatures. The aim of this research is to develop isothermal vitrification methods to ensure that the archival serum samples are stably stored at room temperature for a long time.

lyoprotectant matrix

We have developed a lyoprotectant matrix that is used for stabilizing archival serum samples. Serum is pipetted into the wells filled with the lyoprotectant matrix, and dried overnight in a vacuum chamber, which vitrifes the sample making it stable during room temperature storage. The matrix is an electrospun non-woven fibrous material electrospun from a patent pending lyoprotectant solution. The capillary forces help adsorb the serum into the matrix, and dissolve to uniformly load the serum with the lyoprotectants, avoiding clumping.

matrix adsorption

The results of the initial validation studies we have conducted have recently been published [Scientific Reports, 6, 24186, 2016];

Adsorbing/dissolving Lyoprotectant Matrix Technology for Non-cryogenic Storage of Archival Human Sera

 

We are currently in the process of collecting longer term storage results after making some modifications to the existing matrix. Stay tuned!

Some questions we seek answers to:

What happens in a solution when it freezes?

Is the ice phase pure?

Where do the buffer salts, proteins, and cryo-/lyoprotectants go when ice freezes out?

Does it mean all the liquid water in the sample is frozen?

What is the difference of storing a solution at -4oC, -20oC or -120oC?

Does the thawing rate matter?

Does the pre-freeze cooling rate matter?

Does the ice nucleation temperature matter?

What happens when a solution vitrifies?

Role of Excipients on Freeze/Dried Protein Stability

trehalose cake

In freeze-drying processes used to stabilize drugs and therapeutic formulations, it is desired that the structure-forming ingredients (e.g. mannitol) crystallize to avoid collapse of the cake while the lyoprotectants (e.g. trehalose) stay amorphous to protect the ingredients. This is a challenge as it is very difficult to come up with the formulation that would enable this under conditions of low temperatures, crowding etc. Our recent publications in this area, helps establish the guidelines while shedding light on the phenomena at work at the molecular level.

Effects of Excipient Interactions on the State of the Freeze-Concentrate and Protein Stability

Mutual Influence of Mannitol and Trehalose on Crystallization Behavior in Frozen Solutions