Upstream Processing II

• The lecture discusses the nutrient requirements in protein biologics manufacturing, emphasizing the components of culture media for culturing host cells to synthesize recombinant proteins.

In Vivo Environment – Body Fluids

• The extracellular fluid's essential constituents and physical characteristics, control ranges, and nonlethal limits are described in the table.
• Macronutrients are needed in concentrations greater than 10⁻⁴ M.
• Micronutrients (trace elements, vitamins, hormones) are needed in concentrations less than 10⁻⁴ M.

Cell Culture

• Cell culture grows or disperses cell collections under laboratory or industrial conditions.
• Conditions include providing an artificial environment, nutrient solutions, a surface for growth, and ideal temperature, humidity, and gaseous atmosphere conditions
• Culture medium should provide water and nutrients
• Conditions for environmental control include osmolarity, pH, aeration, temperature, and mixing

Water in Biopharmaceutical Products Manufacturing

• In upstream processing, water is a component of the cell culture media.
• In downstream processing, water is used for sterilizing bioreactors, large-scale fermentation/cell culture, protein extraction and purification, and final product formulation
• For industrial manufacturing, Water For Injection (WFI) is generally used, particularly in downstreaming processes
• US Pharmacopeia defines WFI with specific limits for pH (5 to 7), chloride (0.05 mg/L), sulfate (1.0 mg/L), ammonium (0.03 mg/L), and calcium (4.0 mg/L), CO2 (5mg/L), Heavy metals (1.0mg/L), Oxidizable substances (0.8mg/L), Total Dissolved Solids (TDS): 10mg/L

Nutrients in Culture Media

• Low molecular weight nutrients include sugar, amino acids, RNA, DNA precursors, lipids, bulk ions, trace metals, vitamins, and enzyme co-factors.
• High molecular weight nutrients include hydrolyzed proteins, proteins, and serum
• Other additives also included

Nutrients to Build Cellular Components

• Water makes up ~70% of cellular mass.
• Carbohydrates constitute 50% of bacteria's dry weight.
• Nitrogen accounts for 14% of bacteria's dry weight.
• Phosphate comprises 3% of bacteria's dry weight.
• Salts (Mg²⁺, SO₃²⁻, Ca²⁺) constitute 5% of bacteria's dry weight.

Carbon Source from Sugars

• Glucose is commonly used as a carbon source in culture media.
• Other sources: sucrose, galactose, mannose, fructose, ribose, xylose, pyruvate, glycerol, CO₂
• Serve as energy, building blocks for cell wall, and pyruvate acts as an amino acid precursor.
• Lactate can be produced from pyruvate (lactic acid fermentation)
  • Affects pH and osmolality
  • Glucose concentrations in the culture medium range from 1-6 g/L.

Nitrogen Source

• Inorganic nitrogen sources include ammonium salts (NH₄⁺) and nitrates (NO₃⁻) for plants/some bacteria.
• Organic nitrogen source is urea
• L-amino acids are secondary energy sources and building blocks for cellular structures and enzymes.
• Essential amino acids cannot be synthesized by cells/organisms and must be supplied.
• Non-essential amino acids can be synthesized by cells/organisms and are not necessary EXCEPT glutamine
• Hydrolyzed proteins and peptides provide nitrogen.

Nitrogen Source - Glutamine

• Essential for energy, protein synthesis (non-essential but required), and lipid/nucleotide biosynthesis
• Precautions: it's unstable in solution, may cause high ammonia levels and impair cell growth
• Glutaminolysis increases lactate production and pH imbalance.

Nitrogen Source – Hydrolyzed Proteins

• Used as supplements in cell culture
• Tryptone and yeast extracts exemplified in LB broth
• Product of protein hydrolysis from casein, gelatin, meat, soy, egg, lactalbumin and yeast, containing amino acids, peptides, ions, carbohydrates, and vitamins.
• Advantages: inexpensive and suitable nitrogen source and helps to lower serum concentration
• Disadvantages: not chemically defined, lot-to-lot variability, and may not efficiently support cell growth.

Phosphate source

• PO₄³⁻ provides buffering capacity
• Serves as a precursor for DNA, RNA, phospholipids, phosphoproteins, and ATP
• Needed for reversible phosphorylation/dephosphorylation concerning cell proliferation activities.

Salts (Bulk Ions) and Trace Metals

• Main ions: Na+, K+ for membrane potential, Mg2+, Ca2+ for cell adhesion, and Cl, PO43-.
• Important co-factors for some glycosyltransferase enzymes.
• Found in serum normally
• Trace elements include Fe, Mn, Se, Co, Ni, and Zn.
• Trace elements are good for cell growth, with specific requirements for trace elements in some cell lines. Chemically defined, protein-free media contain these components.

Vitamins and Other Enzyme Co-factors

• B vitamins, vitamin C, vitamin A, vitamin D, vitamin E, and vitamin K are included

Culture Medium by Components

• Example is culture medium for mammalian cells
• Lists Description of Components: Inorganic salts, Essential amino acids, Other components, Vitamins and Serum constituents

Proteins

• Added to allow and/or promote cell growth and stimulate cells
• Epidermal growth factor stimulates growth of some cell lines
• Fibronectin, Laminin, and Vitronectin promote attachment of cells
• Insulin promotes glucose uptake.
• Transferrin allows iron transport into cells.

Serum

• Works to added increase effectiveness of chemically defined
• Function is a complex mixture with multiple (some unknown) functions
  • Carrier proteins
  • Hormones and growth factors
  • Protease inhibitor,
  • Nutrients, -Binds toxic substances
• Serum tyes
  • Foetal bovine serum
  • Newborn calf serum
  • Donor calf serum
  • Horse serum
  • Human serum
  • Chicken serum

Serum

• Most common source is Foetal Bovine Serum (FBS)
  • From bovine foetus blood
  • High growth stimulators/low inhibitory factors compared to other sera
• Challenges
  • Variable between lots
  • High cost -Risk of animal-borne virus
  • Protein content removal adds burden
• FDA has concerns about viral contaminants -Avoid animal products
  • Use animal component-free media for cell culture

Serum-Free and Animal Component-Free Media

• Growth factors and hormones may still be important
  • Growth and maintenance factors
  • Transport proteins,
  • hormones
  • Cytokines
• Animal Component-free media:
  • Non-animal products used, like yeast hydrolysate, soy hydrolysate and non-animal lipids
  • Use proteins from non-animal sources like recombinant insulin, or e. coli proteins

Protein-Free Media

• Easy purification due to lack of proteins
• Chemically defined protein-free media replaces protein components with inorganic compounds
  • Reduces lot-to-lot variability
  • Aids in subsequent analysis/optimization of cell culture performance

Cell Culture – Culture Media

• Glucose is present, needs all energy from glucose, and manufactures organic molecules from glucose.
• LB (Luria-Bertani) Broth is culture media
  • 10 g tryptone
  • 5 g yeast extract
  • 10 g NaCl

Cell Cuture - Culture Media

• Dulbecco's Modified Eagle's Medium (DMEM) used to culture Mammilian cells
• Requires proteins and serum to support mammalian cell growth