Skip to main content

Hitachi

Hitachi High-Technologies GLOBAL

Biomedicines are protein-based drugs manufactured by applying recombinant DNA technology or cell culture technology and one of the characteristics is that their raw materials are biological-origin polymers. Some guidance and guidelines have been published for the test and evaluation methods.
"Peptide mapping method," one of the methods to identify biomedicines, is introduced here.
The peptide mapping is a method developed to analyze the properties of proteins, evaluate the identity and stability, and detect the mutation. To compare the elution patterns of LC chromatograms showing many peptide peaks, it is extremely important to ensure that the repeatability for the peak retention times and areas are good.
This time, BSA and an antibody drug (IgG), a typical biomedicine, were used as model samples and the repeatability was evaluated based on the LC analysis of their digests.

Summary of Peptide Mapping Method

In this method, the changes in constituting amino acids are confirmed by comparing the chromatographic patterns after the peptide fragments formed by the chemical or enzymatic digestion of proteins are separated and detected by instruments such as LC.

*
Peptide mapping is a method specified based on the harmonization of three pharmacopoeias (USP, EP, JP) and is described as a reference in the JP (Japanese Pharmacopoeia) 16th edition.

Analysis Example of BSA (Bovine Serum Albumin) Digest

Analysis Conditions

Column HITACHI LaChrom C18 (5 µm) (4.6 mm I.D. x 250 mm)
Eluents A)0.1% TFA/H2O(v/v)
B)0.1% TFA/CH3CN(v/v)
* Gradient
Flow rate 1.0 mL/min
Column temperature 40°C
Detection wavelength UV 215 nm
Injection volume 10 µL

Sample Preparation

*
A dynamic mixer is used.

Peak Retention Time and Peak Area Repeatability (n=6)

Retention Time

Peak No. 1 2 3 4 5
Average 13.579 23.998 30.508 34.488 44.911
SD 0.009 0.012 0.012 0.018 0.012
%RSD 0.06 0.05 0.04 0.05 0.03

Peak Area

Peak No. 1 2 3 4 5
Average 313951 477180 729175 922057 814068
SD 6957 11499 17599 22397 21035
%RSD 2.22 2.41 2.41 2.43 2.58

When the analysis was repeated six times, the repeatability for the retention times (%RSD) was 0.06% or less and that for the peak areas (%RSD) was 2.6% or less, indicating that extremely good repeatabilities were obtained.
Therefore, it was confirmed that the peptide mapping method allows the highly reliable chromatographic pattern analysis.

Structure of Antibody Drug (IgG)

Among biomedicines, many antibody drugs prepared by making use of antibodies (immunoglobulins) are proteins having glycan structures and they are especially drawing attention for their high specificities and usefulness. While there are several types of immunoglobulins, IgG is mainly put to practical use for antibody drugs and it characteristically has a "Y"-shaped four-chain structure. Two each of the H-chain and Lchain are linked by S-S bonds (disulfide bonds) so as to form a Y-shaped heterotetramer. It is also considered that the structures of the glycans, that are linked due to post translational modifications, largely affect the activities of the medicines.

This time, IgG was subjected to a reductive alkylation and then, trypsin-digested. The fragments of the peptide was analyzed by the LC and the repeatability of the peak retention time and areas were evaluated based on the chromatographic patterns.

Analysis Example of IgG Digest

Analysis Conditions

Column HITACHI LaChrom C18(5 µm)(4.6 mm I.D. x 250 mm)
Eluents A)0.1%TFA /H2O(v/v)
B)0.1%TFA/CH3CN(v/v)
*Gradient
Flow rate 1.0 mL/min
Column temperature 40°C
Detection wavelength UV 215 nm
Injection volume 10 µL

Sample Preparation

Peak Retention Time and Peak Area Repeatability (n=6)

Retention Time

Peak No. 1 2 3 4 5 6
Average 10.960 11.934 22.716 26.630 34.979 43.647
SD 0.012 0.011 0.013 0.010 0.013 0.010
%RSD 0.11 0.09 0.06 0.04 0.04 0.02

Peak Area

Peak No. 1 2 3 4 5 6
Average 131165 80680 227672 135253 418818 200076
SD 2547 2585 6966 4474 16010 8164
%RSD 1.94 3.20 3.06 3.31 3.82 4.08

When the analysis was repeated six times, the repeatability for the peak retention times (%RSD) was 0.1% or less and that for the peak areas was 4.1% or less, indicating that extremely good repeatabilities were obtained. It was shown that the highly reliable analysis of chromatographic patterns is possible even for complex peptides such as digested IgG.

NOTE:
These data are an example of measurement; the individual values cannot be guaranteed.
The system is for research use only, and is not intended for any animal or human therapeutic or diagnostic use.

Donwload Adobe Reader
In order to read a PDF file, you need to have Adobe® Reader®