Although the ordinary state of most atoms and molecules is neutral, in some instances a charge can occur. These charges cause different interactions between molecules that can be taken advantage of in manufacturing.
In an ordinary atom, the number for protons equals the number of electrons so there is no electric charge. An Ion refers to atoms that have an imbalance and therefore have either a positive or a negative charge.
Cations are positively charged ions and have more protons than electrons by losing electrons. Anions are negatively charged ions and have more electrons than protons by gaining electrons.
The active ingredients in many of our biologics products are proteins produced from cells. Proteins are chains of amino acids strung together. These chains of amino acids fold together to form a pattern.
Because each amino acid has different structure, some inherently have a charge. When proteins fold, it leaves patches of positive and negative charges on the surface. Depending on the pH, these charges can be manipulated to be more positive or negative. We can take advantage of these charges to purify our products.
Proteins can also have a charge depending on their environment. Changing the pH is a common tactic used to change the charge. A low pH environment corresponds to a very high concentration of protons, while a high pH environment corresponds to a low concentration of protons.
The isoelectric point (pI) is the pH at which there is no net charge on a molecule. When the pH is increased above the pI for a protein, it will become predominantly negatively charged. The opposite would happen if the pH was lowered below the pI, causing a net positive charge.
Ion exchange chromatography is commonly used to separate proteins because they contain charged functional groups. It uses the charges of the molecules to bind the target protein or impurities to a column to purify the product. This process is frequently used in biologics purification, like in Skyrizi.
The stationary phase consists of tiny beads which are attached to chemicals possessing a charge. As the mobile phase (liquid solution containing the mixture of molecules to be separated) flows through the column, oppositely charged particles will stick to the column. Molecules that are neutral or similarly charged will flow through the column.
To detach the molecules that bound to the resin, the pH is either increased or decreased depending on what resin is used for the stationary phase.
Bind and Elute chromatography is when the target molecule binds to the column and is then eluted out after the column is rinsed. In ion exchange, the target molecule would be the opposite charge of the stationary phase and stick to the column.
Flow-through chromatography is when the target molecule flows through the column while other impurities stick to the column. In ion exchange, the target protein would be similarly charged and be repelled away from the charges in the stationary phase, therefore flowing through the column.
In a Cation Exchange Column, the stationary phase is negatively charged and positively charged compounds in the mobile phase will stick to this negative charge. Molecules is the mobile phase that are neutral or negatively charged will flow through the column.
In an Anion Exchange Column (example shown above), the stationary phase is positively charged and negatively charged compounds in the mobile phase will stick to the positive charge. Molecules is the mobile phase that are neutral or positively charged will flow through the column.
