L3 - BIOproduction and BIOtechnologies for Health (BIO²S)

In the field of health biotechnology, recombinant proteins can be produced in different cell types, known as expression systems, which can be prokaryotic or eukaryotic. This is a mature and attractive technological field with high employability. Indeed, the bioproduction of biomedicines (proteins, monoclonal antibodies) represents a major therapeutic challenge in humans. Before designing any biological drug involving a bioproduction step, it is essential to understand the different eukaryotic and prokaryotic expression systems used in biotechnology. 

This course unit includes interactive lectures, presentations by professionals, and practical work during which the knowledge acquired in class will be put into practice: cell culture, production clone controls, recombinant protein production in eukaryotic systems, and production controls. 

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English tutorial for students in the "L3 Bioproduction and Biotechnology for Health" program, aimed at developing professional autonomy in English.

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The main objective of this course unit is to enable students/learners to acquire theoretical and practical knowledge in molecular engineering. That is, to be able to integrate a gene coding for a molecule of interest and express it in a suitable organism. This corresponds to the preparation stage of bioproduction.   

In order to produce a molecule of interest, it is necessary to first understand the mechanisms and molecular tools behind a modified organism (prokaryotic and/or eukaryotic). Knowledge of the enzymes involved in genetic modification and the verification/validation of this modification through vectorization will be covered. Extraction, purification, and expression methods will be covered in practical work. In addition, a comprehensive overview of the importance of molecular engineering in the field of health will be presented.

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This EU incorporates teaching that covers several disciplinary fields and addresses the different methodological approaches used in the industrial bioproduction of a biomedicine. 

Biologics are healthcare products that must meet strictly regulated health control standards for production areas and final product quality control. In order to optimize production times and costs, statistical and mathematical methods are used to model bioprocesses.  

The objectives of this EU are therefore to present:  

- The development cycle of a biopharmaceutical product and the good manufacturing practices imposed by regulations  

- Good laboratory practices necessary to ensure sanitary and secure control of production areas, as well as to ensure the traceability and integrity of experimental data. 

- Quality control of biopharmaceuticals  

- Biostatistical methods and statistical analysis tools (software) for data from experiments 

- Bioinformatics (databases, OMICS, big data processing, etc.) for the characterization of a biomedicine 

- Modeling bioprocesses using the design of experiments method to optimize the production of biopharmaceuticals 

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Immunotechnology is a field of biotechnology that uses knowledge of fundamental immunology to develop methods and technologies for therapeutic, diagnostic, or analytical purposes. This course unit will provide an understanding of how immunotechnology can be used at different stages of the bioproduction of a biomedicine, from the research and development stage to the quality control stage. 

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This teaching unit (UE) is part of block 2 of the skills targeted by L3 Bio²S, namely "Preparing, designing, and conducting a bioproduction process."

USP or upstream processing refers to the early stages of the production chain for a biopharmaceutical product. It involves the production of cells (cell therapy) and products (proteins, vaccines, monoclonal antibodies, etc.) in bioreactors.

The program covers:

1/ Knowledge of equipment: bioreactors ranging from laboratory to pilot or industrial scale, traditional systems such as single-use systems and their immediate environment (sensors, etc.)

2/ Management of bioproduction processes.

Students will have theoretical and practical knowledge of the structure and function of a bioreactor and how to prepare it for a culture.

After presenting a standard bioreactor (mechanically agitated and aerated tank), the different scales and the concept of scale-up will be discussed, as well as other types of bioreactors (bubble column, airlift, etc.).

Next, all the preparatory steps prior to culture will be described and carried out both in the form of tTD and during practical work on the Bioproduction teaching platform. This part will cover: the preparation of culture media and the adaptation of their composition, assembly, verification, calibration of probes and the bioreactor, sterilization, implementation of an aseptic sampling system, and preparation of the inoculum (cell banks will be covered in EU 1.3 - Recombinant protein expression systems).

Next, the focus will be on "How to conduct and implement a culture in a bioreactor."

In this section, we will discuss:

- Indicators for monitoring biological reactions and monitoring (offline, near-line, and online techniques) and crop management. The program includes: control loops, an introduction to PAT (Process Analytical Technology) and its integration with quality procedures (e.g., implementation of monitoring indicators, control charts, etc.).

- Management of industrial production constraints (in particular limitations due to transfer phenomena such as heat and gases (_O2))

- Different operating modes (batch, fed-batch, continuous): after a general presentation of the different modes, each one is examined in detail. The aim is for students to become involved in an engineering approach to developing viable technical solutions so that they are able to:

o Analyze experimental results

o Use a production planning tool (simulation) on an industrial scale (or pilot-scale experimentation).

o To anticipate the various possible scenarios for feed management for fed-batch and continuous cultures

o To carry out crops during practical work on the educational platform in Bioproduction

This EU is based on three projects that cut across skill blocks 1 (Mobilizing key theories and concepts for bioproduction), 2 (Preparing, designing, and conducting a bioproduction process), and 3 (Communicating about one's work), namely:

Project A: Production and purification of a monoclonal antibody in hybridoma (laboratory scale – screening). Cell culture (preparation of media, cell counting and culture) will be carried out after the producer clone has been checked (UE 1.3).

Project B: Production of a fluorescent recombinant fusion protein in a prokaryotic host in batch mode bioreactors (laboratory scale). Cultivation and monitoring will be carried out after the construction of the producer strains and their characterization on a small scale (UE 1.2). In addition to carrying out the cultivation and analyzing the results obtained, this practical work aims to master the preparation of a bioreactor (calibration, sterilization, etc.). This practical work is also linked to UE 2.1 for the establishment of the experimental plan and to UE 3.1 for the production of reporting documents.

Project C: Production and purification of a fusion protein used as a veterinary vaccine in a prokaryotic host in fed-batch mode bioreactors (pilot scale). Preparation of the fermenter and fed-batch culture from a cell bank of anE. coli strain. Monitoring of the culture (online and offline parameters) and recovery of biomass, calculation of yields. The complete DSP will be carried out in EU 2.3. And batch records/production reports will be produced in conjunction with EU 2.1 and 3.1.

The educational platform for bioproduction:

USP:

- Eukaryotic and prokaryotic cell culture (type II PSM, incubators)

- 4 instrumented benchtop bioreactors (2 to 5 L capacity)

- 1 pilot-scale instrumented bioreactor (20 L usable capacity)

DSP:

- Centrifuge

- High-pressure homogenizer (Emulsiflex, Avestin)

- AKTAPure + preparative GSTPrep affinity column

- Instrumented pilot freeze dryer

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This teaching unit (UE) is part of block 2 of the skills targeted by L3 Bio²S, namely "Preparing, designing, and conducting a bioproduction process."

DSP or DownStream Processing procedures are the downstream procedures in the production chain of a biopharmaceutical. They involve the extraction and purification of the molecule of interest (recombinant protein or monoclonal antibody derived from a hybridoma). This EU will also cover distribution/packaging aspects and related controls.

The program covers:

1) Knowledge of equipment (cell lysis, chromatography, filtration, freeze-drying, etc.)

2) Process management and quality control of recombinant proteins produced

Students will acquire theoretical and practical knowledge of how cell lysis, chromatography, and distribution/packaging equipment works. They will be able to adapt the choice of equipment and processes according to the characteristics of the target protein.

First, students will study the physicochemical properties of the target protein using simple sequence analysis tools. Based on this data and the end use of the protein, students will propose a purification strategy (tutorial).

These elements will be implemented in the practical part of the course, in which students will purify the target protein while performing a series of online samples and checks on the process to ensure traceability and the final quality of the product.

In a second step, students will be required to evaluate the performance of the process and the quality of the active ingredient as well as the finished product by proposing an analytical approach appropriate for therapeutic proteins. They will thus be required to determine production yields, purity levels, the nature of impurities, and confirm the identity of the target protein (TD).

This EU is based on two cross-disciplinary projects covering skill blocks 1 (Mobilizing key theories and concepts for bioproduction), 2 (Preparing, designing, and conducting a bioproduction process), and 3 (Communicating about one's work), namely:

Project A: Production and purification of a monoclonal antibody in hybridoma (laboratory scale – screening). Purification, titration, and testing of the antibody will be carried out following cell culture (UE2.2) and testing of the producing clone (UE 1.3).

Project C: Production and purification of a fusion protein used as a veterinary vaccine in a prokaryotic host in fed-batch bioreactors (pilot scale). Following the practical part of USP (UE.2.2), the biomass obtained is weighed and then lysed (HHP). Emphasis is placed on controlling critical parameters (viscosity, pressure, concentration, etc.), the efficiency of the chosen process (yield, purity), and the storage conditions of the protein produced (stability, sterility, etc.). All results, analyzed critically, may be presented in the form of a batch production report.

The educational platform for bioproduction:

USP:

- Eukaryotic and prokaryotic cell culture (type II PSM, incubators)

- 4 instrumented benchtop bioreactors (2 to 5 L capacity)

- 1 pilot-scale instrumented bioreactor (20 L usable capacity)

DSP:

- Centrifuge

- High-pressure homogenizer (Emulsiflex, Avestin)

- AKTAPure + preparative GSTPrep affinity column

- Instrumented pilot freeze dryer

- SDS-PAGE and Western blot, UV spectroscopy, nanoDSF

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In biotechnology laboratories and companies, scientific, technical, and regulatory documentation is essential for project management, ensuring the reproducibility of experimental results, and complying with biological product manufacturing standards. Knowing how to record, analyze, and communicate information related to the production of a biomolecule makes it possible to document all stages of a bioproduction process and thus guarantee the quality, safety, and efficacy of biopharmaceutical products. 

Through lectures and tutorials, this course unit will present: 

-Tools for conducting scientific and technological monitoring 

-Writing methods for drafting standard operating procedures (SOPs) and experimental protocols.  

-Writing methods for producing detailed reports on experimental results and data analysis (reporting/data communication activities). 

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