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Introduction to Amino Acid Analysis in Biopharmaceuticals

There are 20 naturally occurring proteogenic amino acids; it is these amino acids that are combined together to form the polypeptide chains making up the many different peptides and proteins found across all living organisms.

There are 3 additional non-canonical proteogenic amino acids (selenocysteine, pyrrolysine, N-formyl methionine) that are synthesized into proteins only by very specific translational mechanisms; for example, N-formyl methionine is the first amino acid in prokaryotic synthesized proteins.

The proteogenic amino acids, involved in the synthesis of proteins in vivo, are known as the α-amino acids and are characterized by having both an amine (NH2) and a carboxylic acid (COOH) group attached to the first (α) carbon. The functionality of the individual amino acid residues is determined by a side-chain group (R’) attached to the same α-carbon.

A chain of more than 20-30 amino acids is typically referred to as a polypeptide (Figure 1). The actual order, or sequence, of the amino acids in any given polypeptide, and its associated protein, is referred to as its primary structure and is determined by the gene from which it was transcribed.

Polypeptide chain

Figure 1. Polypeptide chain.

Analysis of amino acids by HPLC can be challenging due to their polar, zwitterionic nature which results in poor solubility near the isoelectric point (Figure 2) and poor UV absorbance characteristics.  Hence, derivatization is employed to reduce their polarity which increases reversed phase HPLC retention and improve UV or fluorescence detection (Figure 3).

Alanine
Figure 2. Alanine
Amino acid derivatization
Figure 3. Amino acid derivatization.

Derivatisation introduces a further step into the analysis cycle, however, it is possible to analyze undervivatized amino acids using reversed phase, ion exchange, ion pair reversed phase, or HILIC modes of chromatography coupled with universal detectors such as ELSD, MS, CAD, or CLND.


Amino Acids in Biotherapeutics

Biotherapeutic proteins are produced using cell culture techniques.  In order that the protein is produced without structural modification and in the highest yield possible the cell culture conditions must be optimized; this includes monitoring changes in the media composition which occur due to cell growth e.g. the uptake of nutrients and release of waste products.  It is important to monitor amino acid content as they are key constituents of the protein as well as important metabolic intermediates.  Amino acid concentration in cell media changes due to consumption of some amino acids and release of others by growing cells.  Monitoring of cell culture media composition is one of the first instances in biotherpeautic protein manufacture where amino acid analysis will be carried out.  Monitoring of these dynamic conditions allows for feeding schedules and media replacement to be optimized.

Following the synthesis of a biotherapetic it is a regulatory requirement that the amino acid composition of mAbs is determined. In general, consolidated and automated methods are widely employed with all methods consisting of four main steps:

1. Hydrolysis

The amino acids are liberated by hydrolysis in a highly acidic environment.  This converts asparagine (Asn) to aspartate (Asp) and glutamine (Gln) to glutamate (Glu) and completely destroys tryptophan (Trp).  Non-proteinogenic amino acids such as norvaline and/or norleucine are added as internal standards.

2. Labelling

The seventeen remaining proteinogenic amino acids are then labelled (derivatized) with a suitable UV/fluorotag - o-phthaldehyde (OPA) for all primary amino acids and Fluorenylmethyloxycarbonyl (FMOC) for the secondary amino acid of proline (Figure x).

3. Separation

Chromatographic separation is then performed on a C18 stationary phase under basic mobile phase conditions using a wide polarity gradient.

4. Detection/Quantitation

The separated amino acids are detected on either a UV or a more sensitive fluorescence detector

Reversed phase analysis of derivatized amino acids is well characterized with the analytes eluting in a predictable manner (Figure 4 and Table 1).

Typical reversed phase chromatogram of a mAb amino acid composition

Figure 4. Typical reversed phase chromatogram of a mAb amino acid composition

Amino Acid Amino Acid Abbreviation Derivative Type
Aspartic acid Asp OPA
Glutamic acid Glu OPA
Asparagine Asn OPA
Serine Ser OPA
Glutamine Gln OPA
Histidine His OPA
Glycine Gly OPA
Threonine Thr OPA
Arginine Arg OPA
Alanine Ala OPA
Tyrosine Tyr OPA
Cystine Cys OPA
Valine Val OPA
Methionine Met OPA
Norvaline* Nva OPA
Tryptophan Trp OPA
Phenylalanine Phe OPA
Isoleucine Ile OPA
Leucine Leu OPA
Lysine Lys OPA
Hydroxyproline Hyp FMOC
Proline Pro FMOC

Table 1. Order of elution for OPA and FMOC derivatives of amino acids.  * Internal standard.

Amino acid analysis can be improved, e.g. the analysis cycle time shortened, by using online, automated derivatization.

To learn more about amino acid analysis watch the February CHROMacademy Essential Guide webcast
UV or MS, Derivatized or Not? Choosing and Optimizing a Workflow for Amino Acid Analysis in Spent Media”.


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Dr. Dawn Watson
 

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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