Bioseparation Engineering 22BT C24

Bioseparation Engineering

• Dr. Dharmalingam K teaches Bioseparation Engineering with course code 22BT C24
• Dr. Dharmalingam K can be found on Google Scholar, Publons, and ORCiD

BSE – 22BT C24 Course Objectives

• Students learn the importance of downstream processing
• Students are taught techniques for cell disruption, solid liquid separation, filtration, and centrifugation
• Principles of membrane-based separations and their applications
• Students are enlightened about chromatographic separations and their role in product purification
• Students study crystallization, drying, and lyophilization

BSE – 22BT C24 Course Outcomes

• Students outline downstream processing of biotechnological process and develop process design for bio products
• Students distinguish techniques of cell disruption and unit operations for separation of bio products
• Compare and contrast membrane separation processes
• Interpret application of chromatographic process for separation of bio products
• Analyze product finishing techniques and case studies of important bio products

Unit I: Role Of Downstream Processing In Biotechnology

• Discusses the role and importance of downstream processing in biotechnological processes
• Includes characterization of biomolecules using CD spectroscopy, UV-Vis, and FTIR of fermentation broths
• Explores the physico-chemical basis of Bio-separations
• Touches on process design criteria for bioproducts and downstream process economics

Unit II: Primary Separation And Recovery Processes

• Focuses on cell disruption methods for intracellular products, including mechanical, chemical, and enzymatic methods
• Separation techniques for removal of insoluble and biomass: flocculation, sedimentation, and centrifugation
• Filtration is explored using theory, equipment-depth filters, plate and frame filters, pressure leaf filters, continuous rotary drum filters, filter media, and filter aids
• Addresses problems related to specific resistance of the cake, time taken for filtration, and compressibility of cake

Unit III: Product Enrichment Operations

• Focuses on membrane-based separations,types of membranes, types of flow (crossflow, tangential flow, mixed flow)
• Types of membrane-based separations (microfiltration, ultrafiltration, dialysis, electro dialysis, reverse osmosis)
• Addresses the design and configuration of membrane separation equipment and applications
• Reviews solution diffusion model, capillary flow model, aqueous two-phase extraction of proteins
• Covers precipitation of proteins with salts and organic solvents and adsorption processes

Unit IV: Product Purification

• Explores chromatographic separations, their principles, and classification
• Offers general descriptions of column chromatography (GC, HPLC; IMAC, bio-affinity chromatography)
• Discusses the design and selection of chromatographic matrices
• Explores large-scale chromatographic separation processes

Unit V: Finishing Techniques

• Introduces pervaporation, supercritical fluid extraction, and electrophoretic separations
• Focuses on final product polishing via crystallization and industrial crystallizers
• Explores drying and industrial dryers, lyophilization, and case studies: citric acid/penicillin with low volume high value product like recombinant proteins

Upstream + Downstream

• Upstream processes involve media hydration, formulation, and cell culture
• Harvest processes involve cell culture bioreactor, harvest collection and harvest
• Downstream processes involve seperation, purification, finishing, bulk storage, final fill and finish

DSP Importance

• Downstream Processing (DSP) involves the recovery of useful products after fermentation or other processes
• The analytical separation aspect of DSP focuses on isolation and purification of bioproducts at varying scales of operation
• DSP techniques requires high cost, where concentration of products are very compared to impurities
• Minimum steps to recover product of interest in pure form and with highest yield in cost effective manner

Why DSP is Needed

• Products are manufactured using various types of reactors, fermenters, or bioreactors
• Principles of product recovery rely on properties: density, distribution coefficient, molecular weight, affinity, charge distribution, and hydrophobicity
• The choice of separation methodology depends on nature of product, as well as quantity and required purity

DSP Steps

• Principles of techniques depends on physiochemical properties of product, media & ingredients used for the recovery process
• Consists of six steps: removal of insoluble, cell disruption, extraction, concentration, purification, and formulation
• A challenge is to efficiently and economically recover a high purity product from a complex mixture

Characterization of Biomolecules

• Each differ in nature and separation
• Most are unstable
• Influenced by pH, temperature, ionic strength, solvent, surfactant, metal ions
• Sensitive to shear and are hydrophobic
• Precent in low concentrations

Properties of Biological Material

• Includes Size, Molecular weight, Diffusivity, Sedimentation coefficient
• Light absorption, Fluorescence, Osmotic Pressure, Electrostatic charge
• Solubility, Partition coefficient, Density and color, Melting and boiling point

The need for Bioseparation

• Enrichment of target product
• Reduction in bulk
• Removal of specific impurities
• Enhancement of product stability
• Achievement of product specification
• Prevention of product degradation
• Prevention of catalysis other than the type desired
• Prevention of catalytic poisoning

Challenges in Bioseparation Engineering

• Low product concentration
• Large number of impurities
• Thermolabile bioproducts
• Narrow operating pH & ionic strength
• Shear sensitivity of bioproducts
• Low solubility of bioproducts in organic solvents
• Stringent quality requirements

Good Bioseparation Process

• Ensures desired purity of product
• Ensures stability of product
• Keeps low cost
• Reproducible
• Scalable
• Meets regulatory guidelines

Range and Characteristics of Bioproducts

• Includes wide range of chemicals
• 3 major categories: market volume, price, and required purity

High value

• Very high purity products produced in grams to kilograms, such as therapeutic proteins, enzymes, factor VIII, interferon, and urokinase
• Diagnostic enzymes like luciferase and glycerophosphate dehydrogenase, human growth hormones, tissue plasminogen activator
• Mabs and insulin, produced in tens or hundreds of kilograms

Bulk Industrial Products

• Includes organic acids, amino acids, ethanol, antibiotics and proteases
• These are produced in hundreds of kilograms to tons in quantity

Circular Dichroism (CD) Spectroscopy

• Uses a spectroscopic technique: it studies optical properties of chiral molecules
• Based on differential absorption of left- and right-circularly polarized light by optically active substances
• Its a powerful tool to analyze protein molecular structures/conformations

CD Instrumentation

• Contains a light source (xenon/deuterium lamp is used)
• Monochromator (filter wheel) used for specific wavelengths of light
• Polarizes unpolarized light with a filter wheel, using lambda/4 phase plate, converts circularly polarized light
• Includes sample holder, holds sample in quartz cuvette, and measures Transmitted light

Principle of CD Spectroscopy

• Chirality: Molecules, where mirror image is not superimposable interacts with right/left circularly polarized light
• DifferentialAbsorption refers to difference in absorbance between right/left circularly polarized light
• The resulting spectrum measures 2nd/3rd Structure of molecules

Advantages of CD Spectroscopy

• Non-destructive, requires little sample, used to analyze protein molecular structures
• Provides rapid insights into structures and conformations
• Can analyze molecules in native, aqueous environment

Limitations of CD Spectroscopy

• Cannot provide structures in atomic level as X-ray crystallography or NMR spectroscopy.
• Less sensitive for large, complex structures.
• Requires optically pure samples to avoid interference from impurities.

UV-Vis Spectroscopy

• Widely used analytical technique in chemistry & material sciences
• Investigates electronic structure of molecules, and determines their concentrations and interactions with each other
• Absorbs light at specific wavelengths and induces electronic transitions (g.s to e.s)
• Relates absorption to the molecules’ structure and environment
• Absorbs between 200 to 400 nm (UV region) and 400 nm to 700 nm (vis region)

UV-Vis Instrumentation and Laws

• Instrumentation uses Deuterium (UV) or Tungsten (Vis) lamp
• Monochromator used for Specific Wavelengths
• Detector converts transmitted light to electrical signal
• Beers law- A =e.cl

Components of UV Spectroscopy

• Components are: UV light source, Monochromator, Chopper, Sample container, Detectors, Amplifier, Recorder
• Deuterium lamp emits light between 200-400 nm (quartz windows) while Tungsten/Halogen filament emits light between 400 – 800nm
• Recorder produces and records all data

UV-Vis Models

• Red shift : presence of an au