Biopharmaceuticals - Preclinical safety evaluation of prophylactic biological products

Concepts and types of biopharmaceuticals

Biological drugs (biopharmaceutics or biopharmaceuticals) are natural biologically active substances and artificially synthesized or semi-synthesized natural substances that are prepared from components such as organisms, biological tissues, or organs, and comprehensively apply the principles and methods of biology, biochemistry, and other disciplines.

Biological drugs mainly include biochemical drugs, biotechnology drugs, and biological products.

Biochemical drugs: generally refer to the basic substances of life extracted from animals, plants and microorganisms, which can also be semi-synthesized by bio-chemistry, or obtained by modern biotechnology, such as amino acids, polypeptides, proteins, enzymes, coenzymes, polysaccharides , nucleotides, lipids and biogenic amines, as well as their derivatives, degradation products and structural modifications of macromolecules.

Biotechnology drugs: refer to drugs derived from biological sources and manufactured using bioengineering technology, including peptides, proteins and their derivatives or products composed of them, such as cytokines, growth factors, monoclonal antibodies, recombinant DNA protein vaccines and endogenous proteins extracted from human tissues.

Biological products: based on the principles of immunology, they are made of microorganisms (bacteria, viruses, rickettsia, and microbial toxins, etc.), animal blood, and tissues, and a class of medicines for diseases that are used to prevent, treat, and diagnose human or animal infections.

Biological drugs are different from chemical drugs

1) Pharmacological and pharmacokinetic characteristics of biological drugs

Most of them are proteins, peptides and nucleic acids, with small dosage, high pharmacological activity and relatively low toxicity.

Poor stability, sensitive to heat, acid, alkali, heavy metal and pH, easily inactivated.

It has a large molecular weight and often exists in the form of polymers. It is difficult to penetrate the epithelial cell layer of the gastrointestinal mucosa, so it is rarely absorbed orally. ;

The biological half-life in the body is short, and it is eliminated from the blood quickly, so the action time in the body is short, and its effect is not fully exerted.

2) The particularity of biopharmaceuticals

1. Incomplete structure confirmation

The activity of biotechnology drugs mainly depends on their amino acid sequence and spatial structure. However, due to their generally large molecular weight and complex spatial structure, existing analysis methods and means cannot completely confirm their chemical structure.

Contents of synthetic peptide structure confirmation:

Amino acid sequence research: Indicate whether the sequence of amino acid connection is correct, often using Edman degradation (ie, determination of N-terminal amino acid), mass spectrometry, nuclear magnetic resonance spectroscopy, and other methods.

Spatial structure research

Biopharmaceutical principles require spatial structure studies, especially for long peptides. If the literature or research shows that a certain polypeptide needs to maintain a certain space to be active, it is necessary to carry out corresponding spatial structure research.

2. Species specificity

Including differences between different races, and differences between humans and animals. There may be differences in the structure or function of the same receptors in different species of animals.

Therefore, biological drugs have different biological activities and even different responses in different species of animals.

3. Versatility

In the same organism, the receptors of biotech drugs may be widely distributed, or target specific cell signaling pathways, which can produce a wide range of pharmacological activities and toxic effects. Therefore, the potential hazards associated with its pharmacological effects should be considered in the application.

For example, certain growth factors EGF, VEGF, and NGF, in addition to promoting the growth of the epidermis, blood vessels, and nerve tissues, are also essential for the normal development of embryos. Blocking these pathways (eg, FGFR antagonists) carries a theoretical risk even if no developmental toxicity is observed.

4. Immunogenicity and immunotoxicity

Immunogenicity: refers to the property of drugs to stimulate the body to form specific antibodies or sensitize lymphocytes. Immunogenicity is a property of the drug itself. Immunogenicity does not necessarily lead to toxicity, but it can affect the objective evaluation of drug toxicity, toxicity, or efficacy.

Immunotoxicity: Refers to the test product causing immunosuppression (infection ↑, tumor ↑) or enhancement (anaphylaxis) or autoimmune response. May be related to pharmacological activity (such as anti-rejection drugs) or not (such as some antineoplastic drugs).

(1) The strength of immunogenicity is one of the decisive factors in the development of biotech drugs.

Therapeutic biological products: the evaluation of their immunogenicity is to investigate the possible impact of the immunogenicity of the drug on the evaluation of drug efficacy and safety.

For example, anti-drug antibodies may neutralize drug activity, affect drug clearance, plasma half-life, and tissue distribution, and alter pharmacodynamics/pharmacokinetics, so that the effects observed in nonclinical studies may not be the true pharmacology and/or effects of the drug or toxic reactions.

Preventive biological products: To evaluate their immunogenicity is to investigate the relationship between the immunogenicity of the drug and its immune protection effect.

(2) Immunogenicity detection is crucial for biopharmaceuticals.

The main purpose of evaluating immunogenicity in non-clinical research is to investigate the immunogenicity of biopharmaceuticals and the possible impact of immunogenicity on safety evaluation.

Preclinical Safety Evaluation of Biopharmaceuticals

(1) Acute toxicity test (single dose):

In principle, all new active ingredients should be subject to this experiment.

In principle, more than two kinds of animals should be selected. The key point is to grasp the poisoning symptoms, their changes over time, and the dose-response relationship. Determination of LD50 is not necessarily required.

(2) Chronic toxicity test (repeated administration)

The dosing cycle must at least reach the clinical planned dosing time.

The dosing frequency should be equal to or greater than the clinical dosing frequency. If the clinical dosing frequency is determined by the half-life, it should also be carried out according to the half-life;

Due to the limitations of solubility, injection times, and the maximum clinically intended dose, the maximum tolerated dose is usually not determined, and the highest dose should be at least 10 times the highest clinically intended dose.

(3) Reproductive toxicity test:

In principle, this test should be implemented for drugs that may be used by pregnant women, pregnancy and lactation.

(4) Mutagenesis test:

Priority should be given to using mammalian cells for mutagenesis experiments

Single peptides or proteins and vaccines that have been shown to be identical to the human form in the natural state do not necessarily require this experiment.

Chromosomal aberrations can occur in both germ cells and somatic cells. Therefore, the chromosomal aberration test can be carried out in these two kinds of cells respectively, generally, bone marrow or peripheral blood cells represent somatic cells, and testicular spermatogonia represent germ cells.

(5) Carcinogenicity test:

Generally standard carcinogenicity experiments (2-year rodents) are inappropriate for biotech drugs; use of transgenic animal models is encouraged.

Pay attention to the evaluation of carcinogenicity of biopharmaceuticals with immunosuppression or promotion of cell proliferation: such as paying attention to histopathological examination and in vitro cell proliferation experiments in repeated drug toxicity.

In case of doubt, consider performing standard carcinogenicity studies (the test substance should be active and non-immunogenic in animals)

(6) Immunogenicity experiment

For repeated administration, it is necessary to check whether the antibody is produced and analyze the influence of the formation of the antibody on the pharmacological effect. Experimental animals that are not easy to produce antibodies should be selected for experiments.

Detect whether there is antibody production, the time of antibody appearance, the number of animals with antibodies, dose relationship, dynamic changes of antibody titer, etc.

Determine whether the antibodies produced are neutralizing antibodies or non-neutralizing antibodies, whether they are related to toxicity, etc.

Neutralizing antibody: refers to an antibody that can neutralize the biological activity of an antigen. For example, after 15 days of continuous administration of rhbFGF, anti-rhbFGF antibodies could be detected in the three rhbFGF dose groups of rats, and this antibody could inhibit rhbFGF to promote the proliferation of NIH3T3 cells.

Non-neutralizing antibodies: may also be of clinical interest, as these antibodies may enhance or reduce drug clearance, thereby altering the drug's half-life, tissue distribution, or exposure time of certain target organs to the drug, which may ultimately affect its activity in vivo and toxicity.

(7) Safety pharmacology experiment

Often combined with single- and multiple-dose toxicity studies; without rodents; with increased renal immune complex testing.

(8) Toxicokinetic study: same as chemical drugs

(9) Local irritation test: local drugs and blood products should be implemented.

(10) Others: such as allergy testing

Preclinical safety evaluation of prophylactic biological products

Attenuated and inactivated pathogens made from viruses, bacteria, or other pathogenic microorganisms through culture and propagation, or immune-rich Prophylaxis, a product that stimulates the body to produce a specific immunological response to prevent a certain disease, is a preventive vaccine.

Preventive biological products (hereinafter referred to as vaccines) refer to preparations that contain antigens and can induce specific active immunity in the human body, which can protect the body from damage caused by infectious agents, toxins, and antigenic substances caused by infectious agents. The safety evaluation of vaccines runs through non-clinical trials, clinical trials and post-marketing evaluations. It includes a series of processes such as control of raw and auxiliary materials, production technology and process, verification of physical and chemical properties and biological properties, animal safety evaluation, clinical safety evaluation, and post-marketing adverse reaction monitoring. This article applies to the preclinical animal safety evaluation of vaccines. The main purpose of the preclinical animal safety evaluation is to investigate the safety of the vaccine through relevant animals, including the impact on the immune organs and other toxic target organs, the reversibility of toxicity, and clinically relevant parameters, and to predict the safety of vaccines in large-scale populations. Adverse reactions that may occur when used in clinical trials, reduce the risks borne by clinical trial subjects and clinical users and provide a basis for the formulation of clinical trial protocols. Toxic reactions that may be caused by vaccines mainly include direct damage to the body caused by the components of the product itself as toxic substances, immune-related toxicity caused by the induction of the immune system, and toxicity caused by pollutants and residual impurities. Since the vaccine works by inducing the immune system to produce antibodies and/or effector T cells, its most important potential toxicity comes from the toxicity related to the immune system, and the methods of conventional drug safety evaluation are not fully applicable to vaccines.

Related animals

Animals of relevant species or strains should be selected for the preclinical safety evaluation of vaccines. Ideal related animals should meet the following conditions: ①Sensitivity to the infectious agent or toxin prevented by the vaccine; ②The immune system is similar to that of the human body, and the immune response is the same or similar to that of the human body after vaccination; ③Sensitive to the inherent toxicity of the product components themselves; ④ There are a large amount of historical control data, and according to the historical control data, it can be judged that the abnormalities in the test are scattered spontaneous lesions in animals or toxic reactions related to vaccines. Since the developmental status of immune organs in juvenile animals is not known, and its correlation or difference with infants or children is unknown, it is currently not recommended to use only juvenile animals for toxicology studies of vaccines intended for use in infants and children. In the preclinical safety evaluation of vaccines, the selection of animal species or strains should have a reasonable and scientific basis.

Immunotoxicity

The immunotoxicity of vaccines is the focus of preclinical research, mainly including hypersensitivity and autoimmunity. If the vaccine is found to have an impact on the immune system (immune organs, immune tissues and immune cells) in toxicology tests, targeted studies on immune function and immunopathology should be carried out. The similarity between vaccine epitopes and host molecules may lead to host autoimmunity, and the pathological consequences of autoimmunity include direct tissue damage, deposition of immune complexes that activate complement, or stimulation of target organ function. Since the current animal experiments cannot accurately predict human autoimmune diseases caused by foreign substances, in the early stage of vaccine development, the possibility of vaccines causing human autoimmunity should be avoided theoretically as much as possible.