The important parts of early drug development
The important parts of early drug development. As we all know, there are many biotherapeutic drug candidates, but there are few drug candidates that can enter the commercial production stage.
Many drugs have died in the early stage of efficacy or safety, and many drugs cannot enter the clinic due to their stability, aggregation and other reasons. Therefore, it is very important to adopt effective strategies and techniques to evaluate, discover and solve problems early. .
In response, the Lonza platform has developed different evaluation tools to evaluate early drug identification and process development stages, such as manufacturability assessment, immunogenicity assessment and protein engineering ( Protein Engineering). In addition, cell line construction is also a very important part of the drug development stage. Let’s learn about these four aspects together.
Figure 1. Developability Assessment Toolbox
Manufacturability assessment mainly focuses on the chemical degradation pathways of drugs, post-translational modifications (PTMs), including deamidation, oxidation and glycosylation, and potential physical stability issues.
1) PTMs affect the binding affinity, function and safety of the drug, and usually affect the biological activity and stability of the molecule. For example, deamidation, oxidation (Trp), isomerization in the CDR region, deamidation, oxidation (Met), isomerization in the Framework region, Asp-Pro hydrolysis sites, cleavage sites, aspartic acid isoforms Early identification and evaluation of conformation sites, oxidation sites, non-standard or unpaired cysteine, N&O glycosylation sites, lysine glycation, etc. save time for later development.
2) In terms of physical stability, the most significant problem is protein aggregation. Aggregates can occur at different stages in the development of biopharmaceuticals, and they can also be expressed in different ways. For example, low host viability, low production, the presence of inclusion bodies, and the formation of milky white solutions or precipitates are all obvious signs of stability problems. Lonza uses the In silico aggregation assessment tool to help predict. This model uses sequence and structural algorithms to identify units with aggregation potential, exposing the aggregation problems that may occur in the later stage of the protein. We can know this risk in advance and solve it.
Immunogenicity Assessment (Immunogenicity Assessment) is also part of the developability assessment. Immunogenicity refers to the product’s ability to inhibit the immune response. Immunogenicity issues can reduce drug efficacy, endanger PK/PD and safety, and in some cases may cause serious side effects, so early evaluation is required. The FDA has made a statement that the organizer needs to provide an immunogenicity risk report and the basic principles of the test when applying for the IND. Lonza has developed very effective tools for this evaluation, “Epibase® in silico”, “Epibase® in vitro” and “Humanization”
Figure 2. An overview of Lonza’s immunology platform
1) “Epibase® in silico” uses a “structural bioinformatics” method to find T cell epitopes in the target protein that may trigger a potential immune response. “Epibase® in silico” contains the latest experimental affinity of peptides and HLA receptors As well as the latest 3D structural features of HLA receptors, candidate sequences can be ranked quickly and effectively. This tool can be used in the identification of T cell epitopes, immunogenicity analysis and protein lead comparison, the screening of low immunogenic protein therapeutic drugs, the elimination of T cell epitopes, and immunogenicity risk management documents.
2) “Epibase® in vitro”, this in vitro platform can assess the immunogenicity risk of biotherapeutic proteins by directly detecting the immune response induced by proteins in human peripheral blood mononuclear cells (PBMC), mainly for the assessment of immune response, immune toxicity, Three aspects of innate immune response and adaptive immune response. Epibase® in vitro assays can be used for clinical detection of cell banks, T cell activation assays, cytokine release experiments, dendritic cell activation experiments, B cell analysis, MHC-related peptide proteomics, bioassays, and potency determinations, etc. Experimental analysis, multi-parameter measurement of human primary immune cell activation by flow cytometry, FluoroSpot and Luminex, can be used to characterize the immune response in detail.
It is very important that the mammalian expression platform, cell line, expression vector used in the early development stage are consistent with the later cell line construction and clinical production. This can save the time of switching between different expression systems and cell lines, and its The post-translational modifications can be kept similar to a certain extent. Lonza’s unique Light PathTM protein production platform can provide enough materials to help us from the safety assessment of the discovery stage to the preliminary in vivo function research, screening and sorting of high-quality candidate proteins, so as to select one or Several antibody proteins that meet the requirements for follow-up development.
1) “Light PathTM Host Screen” is used to select the best expression host system for the target protein. GS Xceed® and XS Technologies® expression systems will be used to evaluate the quality, purity and yield of the protein.
2) “Light PathTM Discovery” is mainly used in the early stage of drug development, small-scale, Non-GMP protein and antibody production. It also provides GS Xceed® and XS Technologies® expression systems. The GS Xceed® system takes about 4-12 weeks. The XS Technologies® system takes about 4-8 weeks.
Figure 3. Light PathTM Discovery
Cell Line Construction
We have screened out antibody protein candidates that are leading in all aspects. For commercial production, high-quality engineered cell lines are needed. The reasons for the failure of some biological therapies have nothing to do with their mechanism of action or efficacy, but because There is a problem with the cell line. Cell line robustness issues, changes in process performance caused by unsatisfactory process design, difficulty in expanding the process to commercial scale, and inadequate or inappropriate drug design. Problems in these links may cause delays in regulatory approvals and project phases. Rework increases project cost and time, making it more difficult for the product to enter the later stages. Lonza company provides four different services (mammal platform), as shown below:
Figure 4. Cell line construction program timelines
1) Full Scope CLC, can be customized. Suitable for customers who target high titer. This procedure is also suitable for complex proteins that are difficult to express. Candidate cell lines can be selected at the 16th week after transfection.
2) Light PathTM CLC can be customized to achieve speed by screening fewer clones. It is mainly suitable for the production of standard antibodies and low-risk proteins such as Fc fusion proteins. Candidate cell lines can be selected at 12 weeks after transfection.
3) Ibex® Design. The design of this fixed program guarantees time and quantity. It is suitable for standard monoclonal antibodies or antibodies of similar molecules. DNA can be declared to IND within 12 months. A minimum of 1.5Kg of GMP raw materials can be provided to meet clinical phase 1. And provide Tox materials within 9 months.
The above-mentioned platform is based on Lonza’s GS (Glutamine synthetase) gene expression system, which mainly includes two host cell lines (GS Xceed® CHOK1SV GS-KO® and CHOK1SV®). Among them, the GS piggyBacTM vector system exerts a powerful function. This system uses A highly active piggyBac transposase delivers the GS Xceed® expression vector to a stable, high gene expression level genomic site. The combination of GS system and piggyBac transposon technology can increase the yield and further improve the stability of the cell line.
Figure 5. GS PiggyBacTM-How it Works
PB transposon system
Transposition is carried out by the “cut-and-paste” mechanism, that is, the transposase cuts the TTAA sequence at the ends of the inverse terminator repeats (ITR) at both ends of the PB transposon, and cuts the transposon from the original locus. Then it is inserted into a new TTAA site in the genome, and the transposable fragment and the transposase gene can be successfully separated after transposition, so that the transposable fragment will not be transposed after being inserted into the genome to achieve the purpose of stable inheritance.
Through the above brief introduction, I have learned that some of the developmental evaluation tools that a drug can choose in the early stage can reduce risks and maximize the opportunity for candidate products to enter the market.
For example, the manufacturability assessment solves the stability and heterogeneity issues that may be caused by the PTMs of the drug, and the immunogenicity assessment solves the risk of drug safety and effectiveness that may be caused by immunogenicity from the early stage of development.
Light The PathTM platform and the cell line construction platform provide high-quality materials and high-yield stable cell lines for drug development. The four escort drug development at different levels.
(source:internet, reference only)