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How to do a good job of biological analysis of peptide drugs?

 Since Robert Bruce Merrifield synthesized the first biologically active peptide in 1953, research on peptide drugs has developed rapidly, and research on peptide drugs targeting multiple receptors has even been carried out internationally. The maturity of peptide synthesis technology has further accelerated the development of peptide drugs and has become one of the international research hotspots. This article gives a brief overview of the current status of the development of peptides as well as the challenges and coping strategies faced in biological analysis.

1 Current status of the peptide drug market

According to QYResearch statistics: the global peptide drug market was approximately US$15.2 billion in 2010 and reached US$28.5 billion in 2018, with a compound annual growth rate of 8.17%, compared to the global pharmaceutical market of approximately US$1.3 trillion in 2018 , The market size of peptide drugs accounted for only 2.19%, and the growth rate of the market size of peptide drugs was about twice that of the overall growth rate of the global drug market. The peptide drug market is expected to grow at a compound annual growth rate of 7.9% in the future. It will reach a market size of 35.8 billion U.S. dollars in 2021, and a market size of 49.5 billion U.S. dollars in 2027. The peptide drug market still has huge room for growth.
See the table below for peptide drugs approved by the U.S. FDA in 2020:

Current status of peptide drug market
Current status of peptide drug market

Most of the peptides developed in the early 1980s did not exceed 10 amino acids in length, and have increased in recent decades, tending to be 40 amino acids or longer. In 2019, oral semaglutide was approved by the U.S. FDA and caused a sensation in the industry. Because of the longer peptide drugs, if they want to achieve oral and intestinal absorption, they must face the challenge of multiple peptidase degradation in the intestine. Oral semaglutide The approval of the industry has undoubtedly played a milestone role in the development of new dosage forms of peptide drugs. In addition to the future development of peptide drugs based on changes and optimization of dosage forms, new peptide drug delivery methods, modified or fusion peptide drugs with extended half-lives will promote the development of such drug molecules. In addition, the development of tumor antigen peptides has also become a hot spot in the current tumor research field.

Medicilon relies on its strong drug innovation technology strength, integrates the company's internal superior resources, and joins forces among departments to build a platform for Medicilon's peptide drug conjugate (PDC) research and development into a technologically with advanced platform, complete supporting facilities and functions and standardized operation. It can form a comprehensive innovative drug research and development platform with integrated innovation capabilities, develop and provide key technologies, and can provide services such as the research and innovative peptide drugs, new peptide dosage forms, and peptide generic drugs.

            Peptide Synthesis

The Medicilon peptide synthesis research team closely focuses on the theme of peptide drug research and is committed to solving the core issues that limit the development and application of peptide drugs. Currently, it has established the synthesis of upstream non-natural amino acid structural units, peptide drug modification, and large-scale synthesis, to a complete system for research on downstream peptide drug signal transduction pathways and mechanisms of action. Through multi-disciplinary cooperation and crossover in chemistry, biology, medicine, pharmacy, etc., the team has achieved outstanding achievements in both basic and applied research.

2 Advantages of peptides as drugs

The rapid development of peptide drugs benefited from their unparalleled advantages: significant activity, strong specificity, good affinity with the receptor, weak toxicity and not easy to accumulate in the body. Compared with protein macromolecular drugs, in addition to peptide vaccines, peptide drugs have relatively small immunogenicity, small doses, higher unit activity, easy synthesis, modification and optimization, high product purity, controllable quality, and rapid Determine the medicinal value.
Advantages of peptide drugs

Polypeptide molecules as drugs also have their disadvantages: peptide drugs have a short half-life and will be quickly eliminated by the body. In order to achieve an effective concentration, frequent administration may be required, which is costly and causes a greater burden on patients’ lives; poor stability and easy to be taken Enzymatic hydrolysis is even sensitive to acid, alkali, high temperature or organic solvents, and is easily inactivated.

3 Challenges of peptide drug bioanalysis

The biological analysis of peptides mainly includes PK pharmacokinetic test, TK toxicokinetic test and anti-drug antibody ADA test. The methods used in the analysis mainly include LC-MS/MS and LBA ligand receptor binding test. In the establishment of analysis methods for peptide drugs, it is precisely because of the various characteristics of peptides that the challenges and difficulties of their biological analysis are caused, mainly in the following aspects:
The molecular weight is small and the half-life is short.
The key reagent for detecting peptides is antibodies. The small molecular weight of polypeptides and the structure is not very complex, which makes the number of polypeptide epitopes limited, which often have hapten-like properties, and the production of antibodies is difficult or the titer is extremely low, which brings great importance to the preparation of key reagents for biological analysis. Challenge. Due to the short half-life of polypeptides, they often appear as rapid elimination in the body, so the sensitivity and lower limit of detection of biological analysis methods have higher requirements.
Mode of administration and dosage.
Among the many ways of administration, intravenous administration is the most bioavailable, and many longer peptide drugs generally have a short half-life in the blood, and they are quickly cleared or degraded after intravenous injection. The low bioavailability of non-intravenous administration poses a challenge to bioanalysis. For example, many hormone drugs are mostly administered subcutaneously, and the drug dose is low, so more sensitive detection methods and lower detection limits are required.
Interference from endogenous counterparts.
When establishing analytical methods for peptide molecules with endogenous counterparts, we need very specific antibody reagents to be able to distinguish endogenous and exogenous molecules, which brings a lot to the preparation of antibodies. Challenge. At the same time, since the endogenous counterparts of polypeptides change dynamically in the body, it is necessary to be able to distinguish and exclude endogenous molecular interference analysis methods.
The impact of stability.
The stability of peptide products is a common problem in the development of biological analysis methods, especially some peptide molecules lacking modification groups or lacking protective groups at both ends of the peptide. Many molecules are fragile and sensitive under high temperature, cryopreservation, shock or acid-base conditions, and even degradation or conformational changes bring challenges to the detailed quality control of biological analysis methods.

4 Coping strategies for peptide drug bioanalysis

In response to the challenge of small molecular weight and difficulty in preparing specific high-titer antibodies, we can adopt an immune strategy that uses macromolecular antigens as carriers to couple with polypeptides, and try different immune adjuvants to stimulate the body’s immune response and produce antibodies. When necessary, the homologous tandem recombination expression of polypeptide antigens can also be considered; when immunizing, select animal species that are far from the polypeptide molecule homology as much as possible for immunization.
For the interference of the endogenous counterparts of peptide molecules, prepare early to provide sufficient time for the preparation of high-quality and selective antibodies, so as to construct a very specific method for exogenous drug molecules. If necessary, try to use internal as much as possible. , Exogenous molecules are separately developed methods to calculate and subtract endogenous molecules; it can also be combined with the understanding of the differences in the structure and properties of the analyzed drug and endogenous counterparts, and targeted strategies can be adopted to eliminate endogenous molecules.
In response to the challenge of poor stability and easy degradation of peptide molecules, the following methods are available to deal with. For example, when sampling, choose anticoagulants or enzyme inhibitors to prevent drug degradation, use glass tubes or add different surfactants to prevent peptide adsorption or aggregation Wait. The buffer formulations used to prepare working solutions or stock solutions are very important in analytical practice. Medicilon Biotechnology’s Pharmaceutical Analysis Department has many experience-based reagent formulations that can be used in practice for everyone to cooperate and try.
To improve the sensitivity of the method, methods can be developed based on different technology platforms. Generally speaking, electrochemiluminescence is easier than chemiluminescence, chemiluminescence is better than fluorescence, and fluorescence is easier to achieve higher sensitivity than ordinary visible light absorption methods. Based on different signal patterns, different molecular labeling methods are selected. The same reagent materials, based on different Assay Format changes can also improve sensitivity. In addition, the increase in sensitivity can also be achieved based on changes in the substrate and the application or combination of signal amplification systems.
The advantages of the detection platform are not absolute. It is particularly important to combine different strategies based on experience and principles.

5 Medicilon Biotechnology Drug Analysis Department

The Pharmaceutical Analysis Department of Medicilon Biotechnology flexibly uses ELISA, ECL, TRFIA, CLIA, IF, IP, CoIP, qPCR, FACS, ELISpot, enzymology and other methods to support cutting-edge biological drugs such as proteins, antibodies (monoclonal antibodies, double Or multispecific antibodies, antibody fragments), ADC, peptides, nucleic acids, vaccines and cell gene therapy and other drugs in the early development, preclinical and clinical stages of PK/TK/Immunogenicity (Total ADA& Nab)/Biomarker&Cytokine and other research evaluations.

Medicilon Biotechnology Drug Analysis Department
Medicilon Biotechnology Drug Analysis Department

Currently, Medicilon has supported multiple ADC drugs involving Her2, Trop2, Muc1 and other targets, as well as EGFR, PCSK9, IL-17A, IL-6, IL-23, VEGF, CD47, TNF-α, CD20, TIGIT and other popular targets and immune checkpoints such as 4-1BB, PD1, PDL-1, CTLA4 and other immune checkpoint monoclonal antibodies or polyclonal drugs, targeting CD19 CAR-T, various fusion proteins, nucleic acids, and many related to diabetes, Research work in different stages of peptide drugs for cardiovascular, bone, tumor and other related diseases.
Medicilon looks forward to cultivating in the field of peptides, empowering more innovative pharmaceutical companies, and relying on R&D capabilities to help the human health industry develop vigorously.

About Medicilon

Medicilon (stock code: 688202) was established in 2004 and is headquartered in Shanghai. It is committed to providing a full range of pre-clinical new drug research services for global pharmaceutical companies, research institutions and scientific researchers. Medicilon’s one-stop comprehensive service helps customers accelerate the development of new drugs with strong project management and more efficient and cost-effective R&D services. The services cover the entire process of pre-clinical new drug research in medicine, including drug discovery, pharmaceutical research and clinical trials. Pre-research. Medicilon has grown up with high-quality customers at home and abroad and provided new drug research and development services to more than 940 customers around the world. Medicilon will continue to base itself on a global perspective, focus on innovation in China, and contribute to human health!

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