Spectrophotometric methodologies used less commonly in late-stage development of proteins, but are very helpful in early development
of biopharmaceuticals, such as CD can be used to study the tertiary structure of proteins. Use of CD does not require the
highly pure concentrated protein solutions needed to prepare protein crystals for X-ray crystallography. A protein's specific
CD spectrum in the near UV region (250-340 nm) is determined by its regular three-dimensional structure in solution. By comparing
the CD spectra of a protein in both a denaturing and non-denaturing solvent, some estimate can be made regarding the conformational
stability of the protein. Because the protein concentration needed to perform CD studies is relatively low, these studies
can be undertaken early in development with small amounts of manually purified protein. Because interpretation of the spectra
is often difficult, in many cases CD spectroscopy analyses are sent to laboratories experienced utilizing these techniques.
Fourier transform infrared spectrometry (FTIR) can also be used to determine the tertiary structure of a protein. FTIR does
not require the protein to be in solution, and it can often be used to support early formulation development for either liquid
or lyophilized proteins.
ELECTROPHORESIS
Electrophoresis is the separation of charged molecules in an electric field. In polyacrylamide electrophoresis (PAGE), the
electric field is formed within the pores of a polyacrylamide gel that are filled with a running buffer. Addition of sodium
dodecyl sulfate (SDS) to the sample preparation buffer as well as to the running buffer is often used to pre-treat the protein
prior to electrophoresis, hence the term SDS-PAGE. In SDS-PAGE, the SDS molecules interact with the protein, unfolding it
and adding multiple charges to the molecule from the associated sulfate groups. Complete unfolding of a protein may require
the addition of a reducing agent as well as the SDS. Proteins migrate through the polyacrylamide gel and are separated according
to their molecular weight in SDS-PAGE.
Another common technique is to run native or non-denaturing PAGE. In native gel electrophoresis, the migration of the protein
through the gel is affected by both the charge and the shape of the protein, as well as the size. While SDS-PAGE is commonly
used to determine molecular weight of proteins, it would be incorrect to use native PAGE for weight determination. Both methods
are used to assess purity of a protein.
Protein is invisible in the gels and must be stained for detection. The most commonly used visualization techniques are silver
and Coomassie blue stains. While silver is more sensitive, the intensity of silver stains is affected by the proteins and
is not linear with the concentration of protein, as is Coomassie blue staining. If the intention is to quantitate the relative
amounts of each protein band, Coomassie blue staining should be used.
In addition to determination of molecular weight, SDS-PAGE is used to examine the presence of aggregates. Samples can be prepared
with and without reducing agent, either mercaptoethanol or dithiothreitol. Comparison of reduced and non-reduced gel patterns
allows the analyst to determine whether the higher molecular weight aggregates seen are due to inter-molecular disulfide bridges.
Additionally SDS-PAGE provides information about the purity of the protein. After scanning Coomassie blue-stained gels and
calculating the area or relative intensity of each band seen in a sample, the percentage of the total protein can be determined.
Most laboratories have scanning software capable of performing both image analysis and quantitation. Many software programs
can also determine the molecular weight using results from the standards run on the same gels.
Isoelectric focusing (IEF) is another electrophoretic separation method. In this method, the polyacrylamide gel or other support
layer also contains a pH gradient. IEF is a powerful method for investigating the charge differences among proteins. In IEF,
each protein migrates through the support layer until it is "trapped" at the point where the pI of the protein is the same
as the pH gradient formed in the support media. At that point, the charge on the protein is 0 and it no longer migrates but
focuses. Separated proteins need to be stained to be visualized. The pI of a protein can be determined in an IEF separation
either by comparison to standards run simultaneously, or by measuring the pH of the band with a special pH electrode. In proteins
with multiple glycosylated forms, it is often difficult to determine the pI because the multiple forms may run as a smear
across the gel. In those cases, the carbohydrates could be enzymatically removed, yielding a single protein form.
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