Indeed, an important stage in formulation development is purification. At the IBC meeting next month, Haripada Maity, Ph.D., principal scientist, formulation development at ImClone Systems, an Eli Lilly subsidiary, will talk about carrying out biophysical analyses to understand and mitigate challenges in downstream purification.
Dr. Maity will present a case study on a monoclonal antibody to demonstrate the “exceptional utility of monitoring unfolding/refolding kinetics over other measurements.”
Dr. Maity explains that although optimization of stability and solubility of a protein at low pH is important for chromatography, “the challenge is to avoid damaging the protein during the purification process and after purification when formulations are brought back to neutral pH.”
“Otherwise, degradation of a formulation during purification can result in poor yield and, ultimately, lead to less profits.”
Dr. Maity will also address the challenges of using conventional methods for optimization and other aspects of biophysical analysis, including conformation stability, structural characterization, physical size, and concentration-dependent protein-protein interactions.
Protein stability is critical and is one aspect of formulation development in which the use of conventional methods may not be enough to understand the effects of various formulation conditions, he maintains.
However, one conventional approach that is invaluable is differential scanning calorimetry (DSC), which provides the formulation scientist with data on conformational stability, which is related to thermodynamic stability, of a protein formulation.
For example, if one wants to determine an appropriate buffer for downstream processing, DSC is a technique that allows one to determine which buffer would be optimal for maintaining protein stability.
“By monitoring the melting temperature, one can understand different buffer systems and pick the best buffer with low pH,” he continues. “Moreover, monitoring the melting temperature is a direct way to understand whether a particular protein is stable or not.”
In addition to monitoring thermodynamic stability with low pH buffers, monitoring the unfolding and refolding kinetics at low pH can provide answers to questions such as, “Has the structure changed?” and “How long does the protein take to undergo a change?” In other words, if one decreases the pH from 7 to 3.5 as a function of time, one would be interested in what structural changes occur and the duration of those changes.
However, the challenge that remains with conventional methods such as DSC is data interpretation. The data can be interpreted in multiple ways. First, a higher melting temperature does not mean higher stability, although, in most cases it is true. Second, DSC can provide two different kinds of data, exothermic and endothermic curves, and the interpretation can be complex.
“Nonetheless,” adds Dr. Maity, “the goal of finding a good buffer is to have a strong correlation between kinetic data and equilibrium stability data."