Quality Control of Amino Acids & Peptides: A Guide

SPPS, purification, and lyophilization typically produce a white powder. This must now be subjected to detailed analysis (quality control, QC) to find answers to the following questions:

• • Is it, in fact, the desired product?
  • How pure is the product?
  • What by-products does it contain?
  • How high is the product content?

Our QC departments have a huge arsenal of equipment and methods at their disposal to analyze our products. The choice of analytical methods also depends on the nature of the product — that is, whether it is an amino acid, a very short peptide, or a longer peptide.

Identity  – Is it the desired product?

If it is the desired product, the measured values should correspond to those in the literature or the values obtained for a reference sample. The following parameters are used to demonstrate identity:

• Melting point
• Optical rotation [α]
• The R_f value as determined by thin-layer chromatography (TLC): This value refers to the ratio of the distance travelled by the substance spot to that travelled by the solvent. This depends on the solvent/mobile phase and is always <1.
• Co-elution with a reference sample by TLC: The mixture of the product and reference sample should give a single spot rather than two spots.
• The molecular weight is determined by mass spectrometry.
• The chemical composition of the product is determined by elemental analysis: The contents of carbon, hydrogen, and nitrogen are measured and compared with calculated values for the molecular formula, e.g., C_x H_y N_z O_w S_v. Oxygen is only determined in exceptional cases. Sulfur can also be measured in-house.
• Spectroscopic methods, e.g., IR, NMR, UV spectroscopy, for amino acid derivatives.
• Co-elution with a reference sample by analytical HPLC (mainly for peptides).
• Amino acid composition: In amino acid analysis, a strong acid is used to break down the peptide into its individual amino acids, which are separated by chromatography and quantified. This method does not provide sequence information.

Purity - How pure is the product?

Purity can be evaluated by various methods:

• TLC for amino acid derivatives, very short peptides, and biochemicals: This is often a valuable method because reference samples of potential impurities (e.g., reactants) can also be analyzed at the same time and a large variety of detection methods is available.
• Analytical high-performance liquid chromatography (analytical HPLC or the faster variant RSLC): This is the standard method for peptides. Purity is typically assessed by UV detection at 210–220 nm, where the area of the main peak in relation to the total area of all peaks reflects the peptide purity.
• Optical purity, to determine the contents of the correct and incorrect enantiomers: This is an important parameter for amino acid derivatives and loaded resins.

Product content  – What by-products does it contain?

In addition to impurities arising during the synthesis, the lyophilized products typically contain trace amounts of residual solvents and/or water. Most peptides also contain trifluoroacetic acid or acetic acid, which is firmly bound to the free N-terminus and basic side-chain functionalities (e.g., Arg, Lys, His) as the salt.

These compounds are not measured by the same methods used to determine the purity of the desired product. In order to calculate the amount of each component needed in a reaction mixture, for example, parameters such as the peptide content and water content must be known. These are evaluated by various techniques:

• Water content: determined by Karl Fischer titration.
• Residual solvents: determined by gas chromatography.
• Acetic acid content: determined by HPLC or ion chromatography.
• Residual trifluoroacetic acid, for our API peptides that are sold as acetate salts: determined by ion chromatography.
• Titration with solutions of acids or bases: for products that are bases or acids, respectively.
• Halide determination: the chloride or bromide content can be determined by titration with silver nitrate solution for products that are sold as the corresponding salts.
• Nitrogen content (from elemental analysis): for peptides, this is a measure of the peptide content.

As such, the “net peptide content” (NPC) is defined as the percentage of peptides relative to non-peptidic material (mostly counterions and moisture). NPC and purity are not equivalent, because the former includes peptidic contaminants. In addition, a low NPC must be expected for peptides containing a large proportion of basic amino acids, even if they are extremely pure, owing to salt formation. This is also the case for hydrophilic peptides, which can absorb considerable amounts of moisture. Both the NPC and the purity must be taken into consideration when preparing solutions of biologically active peptides for assays.

A selection of the obtained results can be found on the Analytical Data Sheet (ADS) or Certificate of Analysis (CofA), which is made available to customers who purchase our products. In the case of GMP products, microbiological analyses are added that are performed in a specialized laboratory at Bachem. For many of our generics, the analyses to be undertaken are specified by the European Pharmacopoeia*. “Ph. Eur.” in the name signifies that a drug substance conforms to the specifications of the pharmacopoeia (e.g., “desmopressin acetate Ph. Eur.”).

Batch-to-batch variability of peptides  – How high is the product content?

Variability in peptide purity

The purity of a peptide can vary from batch to batch, affecting the proportion of the desired product. For example, when a peptide is ordered at 80% purity, the quality can range from 80% to 100%. The variability is more noticeable when requesting lower purity levels. Consequently, quantitative assay results can fluctuate unpredictably, depending on the batch quality.

Peptidic by-products and their effects

Batches with low purity may contain various peptidic by-products, which can differ in both proportion and structure. While some of these impurities might exhibit biological activity, it is not necessarily the same as the activity of the target peptide. In some cases, these by-products can interfere with assays.

Net peptide content and influencing factors

The Net Peptide Content can vary based on several factors, including peptide polarity, lyophilization process, storage conditions, and exposure to humidity. For accurate biological assays, unpurified peptides should not be used, as they may still contain harmful non-peptidic impurities such as small amounts of scavengers.

Handling residual contaminants

Peptides purified through standard methods and lyophilized generally contain only trace amounts of cytotoxic non-peptidic contaminants, like residual solvents and scavengers. However, TFA (Trifluoroacetic acid) cannot be completely removed due to salt formation. If residual TFA is a concern, ordering a more biocompatible salt form of the active peptide may be advisable. Keep in mind that this will require an additional ion exchange step and an adjusted price for the custom peptide.

Recommended peptide purity

Four standard product grades are offered by Bachem (see Table 15), and intermediate purity ranges can be provided on demand. The lower the required level of purity, the lower the price will be. The correlation between purity and price is not linear, however. Efforts and costs for obtaining very pure peptides (97–99%) may increase exponentially.

Table 15: Product grades offered by Bachem and recommended applications