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BIOLOGICAL BUFFERS MAKE PROTEINS HAPPY

Happy Protein-1

What are biological buffers used for?

Biological buffers are used in a wide variety of biopharmaceutical processes involving protein-based drugs.  The use of buffers protects the protein from changes in pH, which can cause denaturation, aggregation, or fragmentation.  In order to maintain the correct environment during the various phases of production, different buffer solutions can be needed for each step.  Most of the purification steps in the downstream processing of biologic API’s (Active Pharmaceutical Ingredient) require buffers to condition, wash or concentrate the product.  Buffers are also used in the isolation and purification processes to promote protein capture and polishing.  They are often used in the filtration process to remove viruses, and can be used for cleaning and disinfecting chromatography matrices and membrane filtration systems.  It is imperative that the buffer solutions be accurate in concentration and be consistent from batch to batch in order to maximize the recovery and yield of the desired protein.

PNG Sartorius 1What is a buffer?

A buffer consists of a weak acid and its conjugate base or a weak base and its conjugate acid.  Weak acids and bases do not dissociate completely in water, but instead exist in solution as an equilibrium of dissociated and undissociated species.  An example of a weak acid is acetic acid.  When it is diluted with water, it exists in solution as acetate ions, hydrogen ions, and undissociated acetic acid all of which are in equilibrium.  Acetic acid can release H+ to neutralize OH and form water.  The acetate ions can react with H+ ions added to the system to produce acetic acid.  Because the three species are constantly adjusting to restore equilibrium, the pH of the system is maintained.

The quality of a buffer is determined by its resistance to changes in pH when strong acids or bases are added.  The ability to neutralize H+ or OH ions is often referred to as buffer capacity and is at its maximum when pH = pKa.  pKa = -log10Ka with Ka representing the acid dissociation constant.  The larger the value of pKa, the weaker the acid.  A weak acid has a pKa value in the range of -2 to 12 in water.

Why is the pKa value important?

Knowing pKa values is important when dealing with systems involving acid-base equilibria in solution.  Many biochemistry applications rely heavily on pKa values.  For example, the pKa values of proteins and amino acid side chains impact the activity of enzymes and the stability of proteins.  Buffer solutions are used to study biochemical reactions at or near the physiological pH of approximately 7.4 for the human body.

Buffer pKa at 25°C Effective pH Range
HEPES, Free Acid 7.48 6.8-8.2
MES, Monohydrate 6.10 5.5-6.7
MOPS 7.20 6.5-7.9
TRIS 8.10 7.0-9.0
TRIS Hydrochloride 8.10 7.0-9.0

Requirements of biological buffers

Most biological buffers in use today were developed by Norman Good and his colleagues back in 1966.  They identified several parameters that characterized the most effective buffers.  Those parameters are:

  • Solubility in water – Most biological reactions occur in aqueous environments so it is important that the buffer is water soluble.
  • Permeability – In order to prevent concentrating of the buffer within the cell or organelles, the buffer should not be able to permeate biological membranes. Zwitterionic buffers, such as MOPS and HEPES, do not pass through biological membranes.  However, TRIS has a relatively high solubility in fat and may permeate membranes.  This is why TRIS is toxic for many mammalian cells in culture.
  • A pKa between 6 and 8 – Since most biochemical experiments have an optimal pH in the range of 6-8, it is important that the biological buffer selected has a pKa close to the pH value. That is when the buffer’s ability to neutralize H+ or OH ions will be at its maximum.
  • Minimal Effect on the Reactions – The buffer components should not interact with the ions involved in the biochemical reactions being explored.
  • Minimally affected by changes in temperature and concentration – The pKa of a biological buffer should be influenced as little as possible by the concentration of the buffer, the temperature and the ion composition of the medium that is used. As a rule, amine buffers such as TRIS, have pKa values that are more sensitive to temperature changes than carboxylic acid buffers.  For example, TRIS buffer prepared at 68°F with a pKa of 8.3, will shift to a pKa of 8.8 when used at 39°F.  Choose a buffer with a pKa value at the mid-point of the pH range of the test system.
  • Minimal interaction between buffers and reaction components – A buffer should not be an enzyme substrate or enzyme inhibitor and should not react with any other components of the chemical reactions being used.
  • Chemical stability – The buffer should not break down under the conditions of its use. It should not oxidize or react with metabolites.
  • UV absorption – Buffers should not absorb UV light at wavelengths greater than 230 nm. Many spectrophotometric measurements are performed in this range, such as the determination of DNA, RNA, and protein concentrations.
  • Purity – Buffers should be easy to purify. Contamination from heavy metals and other species can interfere with delicate biochemical systems.

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