Amino acid analysis (AAA) is a simple technique that has been around for decades but is still relevant, and indeed required, for full structural characterization today. AAA is recommended in the ICH Q6B structural characterization guidelines, which have been adopted by regulatory authorities as defining the expected requirements for structural characterization of biopharmaceutical products.
Despite the simplicity of amino acid analysis, the data generated are key to determining a specific property of the molecule of interest, namely the extinction coefficient, from which concentration determinations can be made for the sample. This has a direct bearing on bioassay work and assessment of potency of the product, which both need to be related back to sample concentration.
When it comes to calculating the extinction coefficient (or extinction factor, ε) of a given sample, the Beer-Lambert Law gives us the relationship between Absorbance (A), concentration (c) and path length (l):
A = ε (mol L-1 cm-1) * c (mol L-1) * l (cm)
This is where amino acid analysis comes in. The technique is used to accurately determine the concentration of a solution through measurement of the molar levels of amino acids determined to be robust within the analysis, in conjunction with measurements of the UV absorbance of the sample analyzed. Taking these data together we are then able to determine the extinction coefficient.
Theoretical extinction coefficients can be determined for any sample by adding up the individual extinction coefficients of the anticipated amino acids in the protein sequence. However, the problem with this approach is that effects of higher order structure are not considered, whereby the relative proximity of amino acids to one another other will influence their ε values. This means that there will be an increasing divergence between calculated and theoretical extinction coefficients as molecular size increases. Regulatory authorities require extinction coefficients to be determined by experiment for this reason.
During amino acid analysis, the sample is prepared to a typical working concentration (e.g. approximately 1 mg/mL) and then the absorbance of this working solution is determined using spectrophotometry (most commonly at 280 nm). This then gives us the absorbance (A) and path length (l) values.
The actual protein concentration is determined by:
It is very important to remember that whilst what is being looked at are all amino acids, they are still unique chemical entities, due to having different side chains with different chemical reactivities and consequently have different susceptibilities under the hydrolysis conditions, with some being more robust than others. Because of the different susceptibilities of the amino acids under hydrolysis conditions and also how they handle in different protein samples, it is important to optimize the reaction conditions and qualify the method when determining the extinction coefficient. This will require multiple runs of the amino acid analysis method and give the best assessment of its fitness for purpose.
Figure 1: Example Amino Acid Standard chromatogram
The nmoles of each recovered amino acid, in combination with the theoretical primary sequence and molecular weight allows us to work out the concentration of the protein.
In theory, only one amino acid concentration is required in order to deduce the protein concentration and calculate the extinction coefficient. However, the same calculation can be performed with every robust amino acid in order to give the greatest accuracy to the measured value.
The utility of amino acid analysis extends beyond just calculating the extinction coefficient of a purely proteinaceous compound. At BioPharmaSpec, we have also successfully employed amino acid analysis on proteins containing non-proteinaceous components, such as glycosylation, RNA conjugates and PEGylated compounds, to give a few examples.
As long as the overall structure, and therefore the relative contribution of each component to the overall mass is known, then the extinction coefficient can be calculated.
As an example, for a PEGylated product that has 60 kDa protein component and a 20 kDa PEGylated component, we know the relative contribution of the protein component (i.e in a 1 mg/mL sample, only 0.75 mg/mL of the sample is proteinaceous) and therefore can extrapolate to find the concentration of both the protein and the product with these additional modifications.
So, while amino acid analysis is in essence a simple technique, it requires a thorough understanding of the chemistries involved and experience in interpreting the data in context of the susceptibilities of individual amino acids to hydrolysis. AAA is still a requirement for full structural characterization and is key in assessing functional data in an activity sense.
This of course extends into the area of biosimilars, where full structural characterization is also key in any claim of biosimilarity. For biosimilars, multiple batches of innovator and biosimilar are compared and this comparison extends to extinction coefficient determination and a comparison of the measured values for this parameter.
Please contact our scientists today to discuss amino acid analysis of your product!
