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Peptide Quality Controls and Assurance
Peptide Quality Controls and Assurance

Providing Quality peptide products for results you can trust...

GMP Peptide Manufacturing


BSI's peptide quality control and assurance processes and management are rigorously controlled and documented at every step of development and production. From the start of each peptide project, robust and discriminatory analytical methods are employed. Rigorous analytical characterization is conducted for the development of a robust manufacturing process to ensure high quality of the product.

BSI has produced several tens of thousands of peptides, and our peptide scientists have acquired advanced skills in all fields of peptide chemistry. With our experience and technical capacity, we can quickly scale up our peptide synthesis to gram or kilogram levels. Our commitment to our clients is to provide technical expertise and assistance throughout our client's projects and to ensure our developments fit with the requirements of our customers.

Biosynthesis Incorporated has the know-how and the required equipment for performing state-of-the-art automated solid-phase peptide synthesis. After synthesis, cleavage and purification of the peptide product “quality control” (QC) of peptides is the final major step in peptide synthesis. QC of peptides ensures that analytical results of peptide analysis are accurate and precise. The quality and purity of synthesized peptides are generally determined by analytical reversed-phase high-performance chromatography (RP-HPLC) followed by ultraviolet (UV) detection at 210 to 220 nm in combination with mass spectrometry. However, for a “Total Quality Assessment” or characterization of peptides useful for a variety of applications additional testing is recommended. For example, to ensure success in various experiments, TFA may need to be removed, or the solubility of hydrophobic peptides may need to be assessed before performing desired experiments.

Peptide Purity - How is the purity of a peptide determined?

The purity of a peptide is the amount of correct peptide in a peptide product. The purity of a peptide is determined via analytical HPLC, often monitored at 214 nm. The final peptide purity is quantified as the area percentage of the detected peak for the target peptide. The peak area of the final purified peptide corresponds to the area of the main peak in relation to the total area of all peaks detected within the chromatogram. For a less pure peptide mixture, this includes all the eluting peaks for the injected material including the peptide of interest plus impurities that absorb at the monitoring wavelength. Mass spectrometry (MS) is used to determine the molecular mass of the peptide.

Peptide Content - What is the Net Peptide Content (NPC)?

The net peptide content refers to the total content of actual peptide present in the final peptide product. The net peptide content is the percentage of total peptide present in the product. The net peptide content can be accessed via amino acid analysis (AAA) and/or by elemental analysis. All lyophilized peptides most likely contain a variable amount of water plus counter ions. Typically, trifluoroacetic acid (TFA) accounts for most of the counter ions unless they were exchanged for example with hydrochloric acid (HCl). The range of the net peptide content is typical between 70 to 90%, but can be lower as well. This characteristic is dependent on the specific sequence of the peptide. For example, peptides that contain many basic amino acids, even when extremely pure, can absorb a large amount of water, and have a low net peptide content.

Absolute Amount of Correct Peptide

The absolute amount of correct peptide in a peptide product is defined by the peptide content and peptide purity. This is usually assessed by RP-HPLC and mass spectrometry. Normally, peptides are delivered as TFA salts as a result of standard RP-HPLC based purification. The answer to many peptide related research question requires information of the absolute peptide quantity.

Different applications will have a need for different criteria or purity. The following table shows a range of different purities needed for specific applications.

Applications Requiring Purity Range of >95 to >98%

  • Quantitative studies
  • NMR studies
  • Crystallography studies
  • Peptides used as reference standards
  • Enzyme-substrate studies
  • Receptor-ligand interaction studies
  • Blocking and competition studies
  • ELISA and RIA
  • In-vivo studies

Applications Requiring Purity Range of >80 to 95%

  • Production of monoclonal antibodies
  • Enzyme-substrate studies
  • Receptor-ligand interaction studies
  • Blocking and competition assays
  • ELISA and RIA
  • In vivo/in vitro studies
  • High-throughput screening
  • Non-quantitative blocking in immune-histochemical (IHC) and Western blot analysis
  • Antibody affinity purification
  • Plate coating for cell attachment

Applications Requiring Purity Range of >70%

  • ELISA standards
  • ELISPOT assays
  • Polyclonal antibody production
  • Determination of the titer of antibodies in standard ELISA

Applications Requiring Crude >50% immunograde

  • Production of polyclonal antibodies
  • Determination of the titer of antibodies in standard ELISA
  • Others...

Can all impurities be removed from synthetic peptides?

A single HPLC-based purification run usually does not allow for the removal of all impurities. Often a few peptide impurities remain. These can be peptides with deletion sequences, truncated sequences, incomplete deprotection, as well as byproducts generated during synthesis or the final cleavage. For the purification of some peptides, especially for the more hydrophobic ones, an orthogonal purification strategy may be needed.

Typically, quality control and assurance of synthesized peptides are determined with the help of analytical RP-HPLC followed by UV detection at 210 to 220 nm in combination with mass spectrometry such as ESI-MS or MALDI-TOF-MS.

Types of Peptide Analysis used during Quality Control

HPLC

Peptide purity is determined via RP-HPLC. The resulting chromatogram allows for the determination of the peptide purity. Calculating the peak areas indicates the number and relative amounts of possible by-products.

Mass Spectrometry

Molecular mass of a peptide can be determined by mass spectrometry. This measurement confirms that the correct product will be delivered to customers. In addition, the mass spectrum shows the main impurities in the purified peptide product, if they are present.

MALDI-TOF-MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) as well as ESI-MS (electrospray ionization) are now routinely used as QC methods.

 

Other Test for the Characterization of Peptide Products

Amino Acid Analysis (AAA)

Description: Amino acid analysis is the gold standard to determine peptide quantities. During amino acid analysis the peptide is hydrolyzed under acidic conditions to release free amino acids. After pre-column derivatization, labeled amino acids are separated by RP-HPLC and detected in a fluorescence detector. This method is to determine the peptide content.

When is it needed: Determination of the amino acid composition in a peptide or protein product. Precise determination of the net peptide content to ensure experimental accuracy and consistency.

Counter-ion Quantification

Description: Counter ions such as TFA or HCL present during peptide synthesis can also be present in the final product. Ion exchange chr omatography is often the method of choice used for the detection and quantification of counter ions in a product.

When is it needed: Removal of TFA in peptides is needed for cellular assays, active pharmaceutical ingredient (API) formulations, as well as for some manufactured peptide products.

Endotoxin Analysis

Description: Endotoxin tests allow testing for bacterial pyrogens such as liposaccharides (LPS) and other pyrogens. The Limulus amoebocytes lysate (LAL) from horseshoe crabs is used for this. Gel-clot, turbidimetric and chromogenic tests allow detection of endotoxins at levels as low as 0.001 EU/ml.

When is it needed: Endotoxins are components of gram-negative bacterial cell walls. It is advised to test for their presence in peptide products if they are used for cellular assays since they can decrease cell viability or cause an immune response.

Moisture Content Analysis

Description: For many products moisture analysis is often a critical part during quality assurance and control. In synthetic peptides, even after a extensive lyophylization step small amounts of water can be present the amount of which depends on the hygroscopic nature of the peptide. In general, the Karl Fisher method is used for moisture analysis of peptides.

When is it needed: The water content in peptides can vary from batch to batch in peptide products. Moisture analysis will help determining the proteins content in peptide preparations needed to ensure accuracy in peptide stock solutions.

Peptide Content Analysis

Description: The peptide content in a peptide based product can be determined using amino acid analysis as well as the analysis of elemental nitrogen.

When is it needed: This test is needed when the accurate peptide content is needed, for example, to prepare exact peptide stock solutions.

pH Testing

Description: For this test peptides are usually dissolved in water or appropriate buffer systems and a pH meter is used to determine the pH value.

When is it needed: Salts as well as the nature of peptide sequences can can influence the pH of peptide solutions. This test is needed to ensure that a peptide solution has the desired pH value.

Solubility Test

Description: For this test peptides are systematically dissolved in different solvents. The resulting peptide concentration in each solution is measured and the solvent that allows the highest amount of peptide to be dissolved is determined. Specifically, hydrophobic and longer peptides may have the need for the selection of special solvents or buffer.

When is it needed: Hydrophobic and longer peptides can have different specific solubilities. This is particularly true for peptides containing tryptophan, isoleucine, leucine, phenylalanine, methionine, valine and tyrosine when part of a peptide sequence.

TFA Counter-Ion Removal

Description: Peptide products purified with standard methods contain TFA salts. Because of this and depending on the peptide sequence various amounts of TFA can be present in product. Ion exchange chromatography among other methods can be used to remove or exchange TFA counter ions.

When is it needed: This removal step is needed when peptides need to be used for cellular assays, cell culture development, active pharmaceutical ingredient (API) formulations, as well as for some manufactured peptide products.