DMPK characteristics of polypeptide drugs and key factors for bioanalysis and detection | Shanghai Zhangjiang Life Science Salon Hongren Pharmaceutical Report sharing

Dr. Huang Jiangeng, Technical Director of DMPK Department of Macro Pharma

In his keynote speech, Dr. Huang discussed the characteristics of peptide drug DMPK and the main difficulties and strategies of its biological analysis in combination with the rich project experience accumulated by Hongren Pharmaceutical, providing certain guidance and reference value for practitioners in related fields.

As a class of biological drugs with high efficiency, low toxicity and high specificity, peptide drugs have their own characteristics in the preparation method, structure confirmation, quality study, pharmacokinetics and other aspects. Due to the differences in their molecular weight and preparation methods, they cannot be simply classified as small molecule chemical drugs or biological products.

With the development of peptide synthesis and production technology, the length of peptide chain is no longer a limiting factor in the development of peptide drugs. Peptide drugs can be classified according to amino acid number, spatial structure, amino acid type and modification type. Among them, linear polypeptides and cyclic polypeptides classified according to spatial structure are the focus of pharmaceutical enterprises.

1. Due to its poor permeability (large polarity), easy to be hydrolyzed by enzymes and other characteristics, peptide substances lead to low oral bioavailability. At present, the industry mainly focuses on the development of injections.

2. With the development of new materials and new preparation technology, new preparation types have gradually emerged, and oral, inhalation and implantation preparations have become a hot spot in the development of peptide drugs, improving the convenience and compliance of medication.

Interpretation:

From the relationship between peptide chemical bond sensitivity and gastrointestinal stability, the relationship between spatial structure and transmembrane absorption, the effect of different excipients or preparation technologies on oral absorption, to the relationship between peptide structure and drug distribution, metabolism, and excretion...... The research on the characteristics of polypeptide ADME continues to deepen, and the core technologies to improve its absorption and disposal continue to emerge.

In the polypeptide drugs entering clinical or preclinical research, the research on inhalation drug delivery, microneedle delivery, nano drug delivery system, etc., is also increasingly mature, with the emergence of new materials and new preparation technology, polypeptide will still be one of the hot research spots in biomedicine.

First, the characteristics of peptide drugs

1, strong biological activity: low dose, low exposure

2, complex structure: molecular weight is usually large (oligopeptide exception), isoelectric point, amide bond stability, structural modification

3, complex biological matrix: endogenous proteins, peptides, cytokines, etc

For specific peptide drugs, there is often a superposition of multiple influencing factors, and it is necessary to systematically optimize the method.

Second, common difficulties in biological analysis of peptide drugs

1,Peptide drugs are easy to produce non-specific adsorption with consumables or instrument systems

2,endogenous interferences, high requirements for chromatographic separation

3,easy to have matrix effect, sample processing difficulty increases

3. Differentiation difficulties in biological analysis of peptide drugs

1. Oligopeptide

The method is less difficult, such as leprerelin protein precipitation method can reach the lower limit of 5pg/mL detection

2. Cyclic polypeptide

It is not easy to produce characteristic product ions

3. Linear peptides

The amide bond hydrolyzes easily

4. Side chain modified polypeptide

It is necessary to take into account the properties of polypeptides and modified side chains, such as polypeptides with long fatty chain hydrocarbons are easy to co-precipitate with proteins

5. protein fusion polypeptide

LC-MS/MS method: Appropriate enzyme digestion conditions should be selected to detect characteristic peptides; LBA method: Select antibodies with good specificity (such as Dulosin)

6. containing acidic/alkaline peptides

Select the appropriate separation consumables (when using SPE); Select the appropriate chromatographic conditions

7. Polypeptide conjugate (PDC)

Multiple detected substances (conjugates, small molecules and/or metabolites, small molecules +Linker, etc.; Couplings may be unstable

8. high molecular weight polypeptide (> 10000 Da)

LC-MS/MS method: The sensitivity of detecting complete peptides is difficult to reach, and suitable enzyme digestion conditions must be selected for detecting characteristic peptides. LBA method: Select antibodies with good specificity.

4. Biological analysis methods of peptide drugs

1. LBA method and LC-MS/MS method

1) LBA method is applied to large molecular weight polypeptides and polypeptide-protein binding

Advantages: high sensitivity, simple operation, high throughput

Disadvantages: Easy to produce false positive results (Cross Reactivity), custom kits require high cost and long cycle time, and cannot quantify multiple compounds at the same time

2) LC-MS/MS method is applied to oligopeptides, polypeptides, PDC, etc

Advantages: good selectivity, can quantify multiple targets to be measured at the same time

Disadvantages: more complex sample processing, easy to produce matrix effect, low detection sensitivity.

Prospects: With the development of enzyme digestion and immunoaffinity extraction, LMS technology will be widely used in the detection of peptides and proteins.

2. Biological analysis strategy of peptide drugs

1) Chromatographic conditions

• Column: Reverse column is the most used, mainly C8 and C18; In a few cases, two-dimensional chromatography was used to improve the separation efficiency. The particle size is usually < 2μm for better sensitivity and selectivity; The pore size is usually 80 A-300 A, and 300 A is better for peptides with high molecular weight.

• Mobile phase: Additives can improve peak shape, sensitivity, residue, etc

2) Sample handling

• Protein precipitation method: simple operation, need to consider protein binding, co-precipitation and other issues; Poor selectivity, easy to produce matrix effect; Suitable for most oligopeptides (e.g., cyclosporine, leprorelin, Goserrelin, etc.)

• Solid phase extraction method: effectively remove endogenous interference (such as endogenous peptides), thereby reducing matrix effects, interference, etc.; It is necessary to select suitable filler solid phase extraction materials based on the physicochemical properties of different peptide components

• Immunoaffinity extraction: The antibody is fixed on the solid phase carrier material with good selectivity; The availability of antibodies and their packing techniques are critical

• Liquid-liquid extraction method: Only for some peptides with small polarity and small molecular weight (such as octreotide)

3) Optimization of mass spectrum conditions

• Ion source selection: ESI source, APCI source is only used in a few cases

• Product ion selection: A product ion larger than the precursor ion m/z is usually selected

• Quality analyzer: QQQ or Qtrap mainly

• Multi-charge characteristics: related to amino acid composition, mobile phase, isoelectric point, etc

• Low ionization efficiency: such as polar, acidic peptides

• Narrow mass range: not suitable for peptides with relatively large molecular weights

• Charge dispersion effect: Additives in the mobile phase (such as m-nitrobenzyl alcohol) can be improved

• It is not easy to produce characteristic product ions: such as cyclic peptides, peptides containing disulfide bonds

4) Improve stability

• Control temperature

• Enzyme inhibitor

• pH regulator

• antioxidant

• Controlled illumination

5) Solve non-specific adsorption

• Replace the low adsorption container with silanized glassware

• Use low adsorption consumables

• Add surfactants (e.g. Tween 80)

• Add protein (e.g. plasma, BSA, etc.)

• Add organic reagents, blocking reagents or structural analogues

• Adjust the pH of the diluted solution

• Try to avoid blow-drying concentration, and add a small amount of DMSO if necessary

• Add appropriate organic solvents to the complex solution

• The pH and ionic strength of the complex solution system were changed

V. Case sharing

1. Study on pharmacokinetics and tissue distribution of peptides in vivo

Specific case:

1) Single and multiple administration of PK in rats and cynomolgus monkeys with innovative peptides

2) Study on tissue distribution of innovative polypeptide drug administration in eye

3) Distribution of PK and tumor tissue in rats with PDC drugs

2. Study on peptide drug DDI

Specific case:

1) Study on metabolic stability of liver S9 of different species of innovative polypeptide

2) Enzyme inhibition of innovative polypeptide CYP450s

3. Peptide drug Plasma Protein Binding (PPB)

Specific case: Semaglutide PPB

PPB evaluation methods for peptide drugs:

1) Balanced dialysis and ultrafiltration, polypeptide drugs are prone to non-specific binding (adsorption)

2) Ultracentrifugation, surface plasmon resonance (SPR) is limited by equipment, and the price is more expensive

3) The dextran charcoal adsorption (DCC) method can be used to study PPB

4. Study on metabolites of peptide drugs

Specific case: Identification of leprerelin metabolites

Main difficulties/concerns:

1) The concentration of drugs and metabolites in the body is low, UV absorption is weak, and is limited by the resolution and sensitivity of mass spectrometry

2) It is difficult for the software to identify the cyclic peptide structure of non-disulfide bond and non-amide group connection, which may cause problems such as missing metabolites or multi-charge identification errors, which can not accurately identify metabolites

3) It is difficult to use UV peak area for semi-quantitative analysis of prototype drugs and metabolites

4) The semi-quantitative analysis of the peak area of mass spectrometry may be interfered by the different ionization efficiency of the mother drug and its metabolites

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About Hongren Biopharmaceutical

Wuhan Hongren Biopharmaceutical Inc.

founded in 2011, is headquartered in Donghu High-tech Zone, Wuhan, with more than 18000m2 of laboratory and office space in Wuhan, Shanghai and Guangzhou. It is a drug research and development technology service CRO enterprise featuring clinical bioanalysis and non-clinical pharmacokinetics. The main business includes clinical bioanalysis, non-clinical pharmacokinetics and pharmacodynamics research, drug and package material analysis, data management and statistical analysis.

More than 480 professional and technical team, equipped with more than 200 instruments related to analysis and testing, Including triple four-bar series mass spectrometer, high resolution mass spectrometer, ICP mass spectrometer, PCR instrument, MSD, Simoa, flow cytometer, ELISPOT, protein sequencer, SEC-MALS, DLS dynamic Light Scatterer, ultra-high performance liquid chromatograph, GMS, etc. The project has rich experience, and has completed more than 1,100 chemical generic drug BE bioassay projects, more than 330 non-clinical pharmacokinetic projects of innovative drugs and more than 200 clinical bioassay projects of innovative drugs, covering various chemical drugs, traditional Chinese medicine and biological products. At present, more than 390 of the projects involved in bioanalysis testing have obtained production approvals or new drug certificates issued by the State Food and Drug Administration. In addition, Hongren Pharmaceutical has passed the laboratory quality assessment certification of the National Health Commission, the capacity certification of the China National Inspection Institute, CNAS 17025 certification, and obtained the biosafety Level II laboratory filing certificate, laboratory animal use license, radiation safety license, and won the title of national specialized and special "Little Giant" enterprise in 2022.