Introduction to Bioprocessing

B99BP Week Upstream bioprocessing

• This course introduces the topics of bioprocessing including bioreactors and fermentation through to downstream processing and product separation.
• The first part of the course (weeks 1-5) gives the necessary background in biological systems, focusing on upstream processes including cell systems, process equipment, and scale-up
• Later weeks will focus on downstream processing requirements for product recovery and integration of both streams to provide economical and environmentally sustainable processes.
• Week 11 looks at sustainability and biorefineries in more detail.
• Weeks 1-5 material is provided as weekly handouts with lecture and tutorial slides and activities.
• Learning outcomes include discussing products from biological systems, understanding cell system requirements and engineering concepts in bioreactor design and describe downstream operations.

Week 0 - Introduction to bioprocessing

• The history of bioprocessing with the range of products, processes, and key areas will be introduced.

Bioprocessing Through The Ages

Pre-1900 Fermented food and drink

• (or in the beginning there was yeast....)
• 7000 - 300 BCE: Yeast was used to brew beer and leaven bread.
• 2000 BCE - 1000 CE: Widespread fermentation of vegetables, pulses, and cereals occurred.
• 1677: Bacteria were first visualized by microscopy (Antoni van Leeuwenhoek).
• 1857: Fermentation and metabolism confirmed yeast's involvement in alcohol production (Louis Pasteur).
• 1877: The term "enzyme" was introduced from Greek in yeast or leavened by Wilhelm Friedrich Kühne.
• 1897: Eduard Buchner showed cell-free yeast extracts could ferment sugar and received the Nobel Prize in Chemistry in 1907 for the work
• 1876 Pasteur published his seminal text on studies on fermentation which looked at the microbial spoilage of beer.
• Pasteur's student also developed a filtration device to remove bacteria from water.

1900-1970s Industrial fermentations

• 1919: Microbial production of citric acid using Aspergillus niger was developed in Belgium.
• 1920s: Acetone-Butanol-Ethanol fermentation by solventogenic clostridia was developed during World War 1 for acetone.
• 1926: James B. Sumner crystallized the first enzyme, urease, and confirmed it was a protein and was awarded the Nobel Prize in Chemistry in 1946.
• 1940s: Large-scale production of penicillin was developed during World War 2.
• 1950s: Amino acid production with use of Corynebacterium glutamicum to produce glutamic acid started.
• 1964: Large scale production of Single cell proteins began.
• 1978: The world's largest bioreactor was ICI for determining Single Cell Protein, SCP production.

1980s-2000

• World War 2 led to the development of commercial scale fermenters with sterile operation because the discovery of penicillin and a demand for its large scale production.
• Industrial fermentations became more common but Single Cell Protein (SCP) became obsolete with availability of cheaper food and growth of oil and gas industry reduced interest in biological production.
• There is a return to SCP for food and green biochemicals.
• 1952: First continuous mammalian cell line established (HeLa human cell line from Henrietta Lacks
• 1957: CHO (Chinese Hamster Ovary) cell line developed
• 1970s: Advent of genetic engineering
• 1975: The First monoclonal antibody generated by Milstein and Köhler at Cambridge.
• Monoclonal antibodies (mAB) are man-made protein antibodies produced using mammalian cell systems and are widely used in cancer treatments.
• 1982: The first licensed drug produced using recombinant DNA technology - Insulin production by E. coli developed by Genentech/Eli Lily
• 1985: Human growth hormone was produced in E. coli and licensed (Protropin produced by Genentech)
• 1986: The First therapeutic monoclonal antibody was approved for use in humans (muromonab-CD3, used as an immunosuppressant in kidney transplants)
• 1986: First recombinant vaccines from yeast was approved (Recombivax HB - Hepatitis B vaccine)
• Before recombinant proteins, biological drugs were generally sourced from animals and humans.
• The advent of monoclonal antibodies has changed the biopharmaceutical industry as biomanufacturing enables production of large biological molecules in microbial and mammalian cell systems.

21st Century

• 2002: Fully-human mAB, was approved for use
• 2003: The Human genome project declared complete
• 2006: The First single-use stirred-tank bioreactor for cell culture occurred.
• 2006: Approval of first plant-made vaccine was given.
• 2007: recombinant production from transgenic animals was approved.
• 2010: The Creation of the first synthetic cell
• Today's cell factories include wild-type, mutated and recombinant bacteria, yeast, fungi, algae, plant, animal, insect, avian, mammalian and stem cells
• The advent of monoclonal antibodies has changed the biopharmaceutical industry and biomanufacturing production of large biological molecules.
• More than 70 monoclonal antibodies were in production for treatment or prevention of Covid-19 while mRNA vaccines based on the mRNA Covid-19 vaccine technology are in development as cancer vaccines.
• Action on climate change has also led to production of industrial chemicals using green processes and a growing population and demand for vegan alternatives has led to a renewed interest in single cell proteins and even cell-based meats and fish.
• Bioprocessing covers production of a wide range of bioproducts influencing industrial processes and biopharmaceuticals.
• Microorganisms involved in bioprocessing range from viruses to stem cells.

Week 1: Upstream processes and microorganisms

• Introduced to bioprocessing areas and microorganisms involved
• Understand upstream bioprocessing scope, discuss historical developments, discuss and compare the basic structure of cells and appreciate the range of organisms and products involved in bioprocessing.
• Upstream bioprocessing stretches from cell system development/selection - including genetic engineering - to fermentation and the start of product separation.
• Processes are integrated, and decisions upstream impact downstream processing, product recovery, purity, and yield.

Biology or Engineering?

• This course we will consider the question of whether you need to be a biologist or engineer to be an expert in bioprocessing.
• Biologists discover the metabolic pathways involved, engineer these to drive production in a particular direction and know about nutrient requirements.
• Engineers concentrate on what concentration to use, how to separate it, scale-up processes, how long it takes, uses, market presence, stream considerations, required energy, and economic factors.
• Bioprocessing requires both biology and engineering.
• It uses living cells or their components e.g. enzymes, to make products.
• A process needs a microbe that can produce the product in high concentrations as well as a design that is achievable and feasible at scale.

Types of products – scale and economics

• Products are classified by chemical class, applications, or cell systems used for production.
• Scale is driven by market demand and economic value.
• Decisions made about scalability should be considered early e.g. if it can be produced in its native host or another cell system if not optimized.
• Cells should be exposed to a range of stresses and their resilience depends on the cell type.

Microbial diversity

• A wide variety of cell systems are used in bioprocessing including prions, viruses, bacteria, archaebacteria and eukaryotic cells.
• Bacteria include Gram positive and negative bacteria (E.coli being most commonly used for biopharmaceuticals), Mycoplasma, Actinomycetes and Cyanobacteria.
• Archaebacteria are interesting sources of thermostable enzymes and methanogens in anaerobic digestion systems.
• Saccharomyces cerevisiae is used for production of fermentative and biopharmaceutical products and has an ability to utilize a wide range of carbon sources including lipids.
• Microalgae are used in biofuel, protein and omega oil production by using CO2 and light for carbon and energy sources.
• Over 70% of biopharmaceuticals are produced using mammalian cells, with Chinese Hamster Ovary (CHO) cell lines being the most common.

Viruses and prions

• Are the smallest microbes that multiply inside other cell types.
• Consist of core genetic material (DNA or RNA), a capsid coat of protein and sometimes a lipoprotein envelope.
• They invade and use the machinery of their host cells to replicate and then lyse the cells to infect.
• Used in bioprocessing for vaccine production and vectors for genetic engineering.
• Gene therapy encompasses any treatment involving delivery of genetic material to modify a gene.
• They can infect the production cells and cause cells to alter or lead to complete shut-down of a facility.
• Prions are infectious agents composed of misfolded protein and can cause diseases by transmitting their misfolded shape.

Bacteria, yeast or mammalian cells?

• A wide range of cells are used in bioprocessing including native or genetically engineered cells producing at high concentrations/recombinant proteins.
• Factors in the choice of microorganism: understanding the structural differences of these cells and how products are made.

Classification and naming

• Phylogeny is the evolutionary relationships between organisms and occurs on a phylogenetic tree and organisms are grouped depending on their structures and genetic sequencing.
• All have a common ancestor termed LUCA, last universal common ancestor.
• Prokaryotes are the bacteria and Archea whereas eukaryotes are the Eukaryota.
• The Linnea naming system is used.

Cell structure and function

• Cells need a barrier to separate the internal and external environment, genetic material to code new cells and materials to transfer in and out - will all impact on cell processing.
• Key Features of the cell includes which cells are larger: bacteria, yeast or mammalian? Which cells will multiply faster: bacteria, yeast or mammalian? Will cell size and shape affect mixing?
• All cells will have:
• A plasma membrane - selective barrier for transport of gases, nutrients and waste
• Cytoplasm and cytosol - fluid inside of cells with subcellular components
• Chromosomes encode genes on DNA
• Ribosomes perform protein synthesis

Summary of the areas influenced by cell

• Influenced by cell structure and function
• Cell growth rate and productivity
• Location of the target product
• Ease of genetic modification
• Stability of cell line
• Resistance to stresses
• Product in the correct form
• Cell form e.g. vegetative or spores; yeast-like or pseudohyphae
• Cell nutrient requirements

Bacteria: Cell structure and use in bioprocessing

• Prokaryotes, lacking a nucleus and are of small size and unicellular and fast growing.
• Used to produce recombinant proteins for biopharmaceutical production but there are disadvantages related to the types and production of proteins in mammalian cells.
• Key features of cells are shown in Figure 10 and cell walls are used to classify them based on their response to Gram staining.
• Gram staining is related to the cell wall structure of the bacteria.

Bacterial cell walls

• Made of peptidoglycan found only in bacteria protecting it from osmotic pressure.
• The Gram stain differentiates the Gram of cells and related to antibiotic resistance.
• Also used to produce a wide range of industrial biotechnology products.
• The Actinomycetes resemble filamentous fungi and are used to produce enzymes and antimicrobials.

Examples of biopharmaceuticals produced by bacteria

• E. coli in particular is engineered although some are produced from native hosts (e.g. botox from Clostridium botulinum.)
• There are some disadvantages to using bacteria for biopharmaceutical production and this is related to the types of proteins which can be produced.

Bacteria as contaminants

• Bacteria including E.coli, Staphylococcus spp. and Streptococcus spp. can also contaminate bioprocessing systems and endotoxins must be avoided where required.
• Cell lines should be tested routinely for contamination.
• Media or cell contamination can occur.

Cell structure and use in bioprocessing - Eukaryotes

• Eukaryotes include animals, plants, algae, fungi and yeast and have distinct organelles in the cytoplasm and are larger than bacteria and divide by mitosis or meiosis.
• The features of Cells may have:
• A Nucleus containing genetic material
• Nucleolus involved in ribosome production and assembles the rRNA with the proteins.
• Nuclear double layer - double membrane enclosing the nucleus with pores.
• Ribosomes - necessary for protein synthesis.

Yeast

• Unicellular forms of fungi can produce recombinant proteins with some post-translational modifications and robust than mammalian cells, with nutritional and fermentation requirements.
• Is widely exploited for production of fermentative and biopharmaceutical products, its genome has been sequenced, and wide genetic engineering tools are available for modification, with varying levels of carbon utilization.

Fungi

• Belong to the Kingdom Fungi but are separate because they are unicellular.
• Multicellular and grow as hyphae or mycelia and produce antibiotics, citric acid and other organic acids and single cell protein, but some secrete high viscosity.

Mammalian cells

• Cell lines are used to produce over 70% of biopharmaceuticals but cells are larger, have no cell walls and slow growing.
• These also have multiple and complex media requirements and releases toxic metabolites into cell growth media.
• Over time cells are unstable.

Human cell lines

• Can produce human-like proteins with the correct posttranslational modifications. Human embryonic kidney cells) and cells are the most common.
• Can grow in suspension serum-free and been approved since the 2000s but can lead to transferring human viruses