Common problems and solutions for bioanalysis of polypeptide drugs based on LC-MS detection platform

The biological analysis of polypeptide mainly includes PK pharmacokinetic test, TK pharmacokinetic test and ADA test. The methods used in the analysis mainly include LC-MS/MS and LBA ligand receptor binding tests. In the establishment of analysis methods for polypeptide drugs, it is precisely because of various characteristics of polypeptides that the challenges and difficulties based on LC-MS biological analysis are caused. Common problems and solutions are as follows:

1. Adsorption problem

Depending on the amino acid side chain of the compound and the state of charge it presents, polypeptides tend to be hydrophobic, and their dot states vary depending on the pH of the environment.

---Solution idea

1) To increase the solubility of the substance to be measured, organic reagents, acids, bases, salts, etc. can be added to change the solvent environment.

2) In the sample preparation process, such as targeted selection of plastic or glass tubes.

3) Add competing reagents, such as plasma, protein-rich substances such as BSA, and surface active reagents.

2. Residual effect

Adsorption usually results in residual and peak trailing.

---Solution idea

1) Common strategies for solving small molecule residues are worth trying for polypeptides and proteins. The higher temperature of the autosampler helps to avoid residue (but this is also the case for column separation when considering stability).

2) Residue removal during gradient elution, continuous injection of high organic phase is not as good as cyclic injection of high organic phase and low organic phase.

3. Stability problem

It is mainly the degradation of polypeptides and proteins under the action of enzymes in biological samples, which mainly occurs in the R functional group of amino acids, including oxidation, reduction, deamidation, and spatial structure change.

---Solution idea

1) By controlling the lower temperature.

2) Add specific enzyme inhibitors (if the object is not clear, the inhibitor combination can be selected).

4. Extract questions

The extraction idea of peptides and proteins needs to be different from that of small molecules, because the traditional extraction method is mostly around the removal of proteins, and in this scenario, proteins and peptides are the target objects.

---Solution idea

1) Due to the large molecular weight of the target, the protein precipitation method may lead to co-precipitation, so it is necessary to pay attention to the selection of precipitating agents. Liquid-liquid extraction, due to the "electric-rich" nature of peptides and proteins, is also often not suitable.

2) SPE, pay attention to the selection of fillers with different pore sizes according to the molecular particles of the substance to be measured. The larger the molecular weight, the larger the pore size of the filler. Ion-exchange SPE is mostly used for the extraction of peptides and proteins.

3) Enhanced retention or enhanced response through derivatization. Common derivatization sites include LYS, TRP and CYS, and the N and C sides are also optional sites.

5. Separation issues

The molecular structure and molecular weight of peptides and proteins affect the selection of chromatographic columns.

---Solution idea

1) Large-aperture chromatography columns and C4 and C8 fillers are mostly used for the analysis of objects with molecular weight greater than 10000. In addition, C18 is still relatively common for the analysis of peptides and proteins.

2) The addition of TFA as a mobile phase reagent can improve peak type (acid) and enhance retention (ion pair reagent), but attention should be paid to the inhibition of mass spectrum signal.

6. Ionization

The ionization problem of polypeptides and proteins mainly focuses on the reduction of signal due to the diversity of their charged states.

---Solution idea

1) Adding different alcohol reagents such as DMSO, isopropyl alcohol, acetone, etc., may have an effect on the charge distribution. Mobile phase composition, proportion, additives, and even the diameter of the spray needle can all affect the charge distribution.

2) APCI model can also be used for the detection of peptide substances under appropriate conditions.