Bioseparations Exam #1 Study Guide

1. Classification of Bioproducts
a. Definition

 Bioproducts: Products derived from biological sources or manufactured using biological

systems (e.g., microorganisms, plant or animal cells)

b. Classes and Examples

Small Molecules (<1 kDa)

 Examples: Amino acids, antibiotics, vitamins, organic acids

Large Molecules (1-1000 kDa)

 Examples: Proteins, enzymes, antibodies, nucleic acids

Particles (>1000 kDa)

 Examples: Cells, viruses, organelles, inclusion bodies

i. Primary vs. Secondary Metabolites

Primary Metabolites Secondary Metabolites

Essential for growth and reproduction Not essential for growth
Produced during exponential growth phase Usually produced during stationary phase
Examples: amino acids, nucleotides, lipids, Examples: antibiotics, pigments, toxins,
carbohydrates alkaloids

ii. Structural Differences and Impact on Bioseparations

 Small molecules: Generally stable, less sensitive to process conditions, can withstand
harsh separation methods
 Large molecules (proteins, nucleic acids): Often sensitive to temperature, pH, shear,
and solvents; require gentler separation techniques
 Particles: Most sensitive to shear forces and require specialized handling techniques

c. Macromolecules, Monomers, and Basic Properties

Proteins

 Monomer units: Amino acids

  Properties: Amphoteric, sensitive to pH and temperature, complex 3D structure

Nucleic acids

 Monomer units: Nucleotides

 Properties: Negatively charged, sensitive to enzymes, UV light

Polysaccharides

 Monomer units: Monosaccharides

 Properties: Often water-soluble, can form gels, variable charge

Lipids

 Monomer units: Fatty acids, glycerol

 Properties: Hydrophobic, form micelles/bilayers in water

d. Protein Properties

i. Protein Structure

 Primary structure: Linear sequence of amino acids connected by peptide bonds

o Stabilized by covalent peptide bonds
 Secondary structure: Regular local structures (α-helices, β-sheets)
o Stabilized by hydrogen bonds between backbone atoms
 Tertiary structure: Overall 3D folding of a single polypeptide chain
o Stabilized by hydrophobic interactions, hydrogen bonds, ionic bonds,
disulfide bridges
 Quaternary structure: Assembly of multiple folded protein subunits
o Stabilized by same forces as tertiary structure plus interactions between
subunits

ii. Protein Folding

 Driven by minimizing free energy

 Hydrophobic residues tend to fold inward (hydrophobic core)
 Hydrophilic residues tend to face outward
 Surface properties impact:
o Solubility
o Charge-based separations (ion exchange)
o Hydrophobic interaction separations
o Affinity-based separations

iii. Protein Denaturation

 Factors that must be controlled during bioseparation:

 o Temperature (avoid excessive heat)
o pH (maintain within stable range)
o Organic solvents (can disrupt hydrophobic interactions)
o Chaotropic agents (urea, guanidinium)
o Mechanical shear forces
o Oxidizing/reducing agents (affect disulfide bonds)
o Surface interactions (air-liquid interfaces)

iv. Monoclonal Antibodies

 Y-shaped glycoproteins (~150 kDa)

 Highly specific binding to target antigens
 Important characteristics:
o High specificity and affinity
o Two identical antigen-binding sites (Fab regions)
o Constant region (Fc) can be used for purification
o Glycosylation patterns affect stability and function
o Relatively stable in neutral pH conditions
o Sensitive to aggregation during processing

2. Stages of Downstream Processing

Stage 1: Solid-Liquid Separation

 Remove solids (cells, cell debris) from liquid phase containing product

Stage 2: Product Isolation

 Separate product from bulk solution components, concentrate

Stage 3: Product Purification

 Remove similar contaminants, achieve high purity

Stage 4: Product Polishing

 Remove trace impurities, ensure final product quality

Stage 5: Formulation

 Prepare final product in suitable form for storage and use

3. Main Challenges for Separation of Bioproducts

 Low concentrations in starting material
  Complex mixtures with similar contaminants
 Sensitivity to processing conditions (temperature, pH, shear)
 Product instability (denaturation, aggregation, degradation)
 Biological activity must be maintained throughout
 Scale-up challenges
 Regulatory requirements for purity and safety

4. General Strategies and Guidelines for Bioseparation

Process Development
 Start with gentlest methods, progress to more selective ones
 Minimize number of steps to improve overall yield
 Consider product stability at each step
 Exploit largest differences between product and contaminants
 Consider scale-up requirements early
 Develop analytical methods in parallel
 Design for reproducibility and robustness
 Balance purity, yield, and cost requirements

5. Bioseparation Processes for Different Product Locations

a. Product is Cells

 Cultivation → Solid-liquid separation (centrifugation/filtration) → Cell concentration →

Washing → Formulation
 Minimal processing, focus on maintaining cell viability

b. Intracellular Product

 Cultivation → Harvest → Cell disruption → Debris removal → Isolation → Purification

→ Polishing → Formulation
 Requires cell lysis step and management of cellular contaminants

c. Extracellular Product

 Cultivation → Solid-liquid separation → Isolation → Purification → Polishing →

Formulation
 No cell disruption needed, but typically lower initial concentration
6. Key Considerations for Bioseparation
Consideration Impact on Process Design
Intracellular vs. Determines need for cell disruption steps
Extracellular
Product Stability Influences process conditions, step order, and speed
Viscosity Affects filtration, centrifugation, and mass transfer rates
Contaminants Determines selection of separation techniques
Product Specifications Sets requirements for final purity and quality

7. Properties Exploited for Isolation and Purification

Property Separation Techniques

Size Filtration, ultrafiltration, gel filtration, dialysis
Charge Ion exchange chromatography, electrophoresis, isoelectric focusing
Hydrophobicity Hydrophobic interaction chromatography, reversed-phase
chromatography
Solubility Precipitation, crystallization, salting out
Biospecific Affinity chromatography, immunoprecipitation
Interactions
Size Filtration, ultrafiltration, gel filtration, dialysis

8. Process and Product Quality Parameters

a. Specific Activity

 Definition: Activity per unit mass of total protein

 Calculation: Specific Activity = Total Activity (units) / Total mass/protein (mg)
 Units: Units/mg protein

b. Concentration Factor

 Definition: Ratio of final concentration to initial concentration

 Calculation: CF = Final Concentration / Initial Concentration
 Dimensionless

c. Purity

 Mass-based: Mass of desired product / Total mass

  Activity-based: Activity of desired product / Total activity of all components
 Mass (protein): amt of protein product / amt of total protein

d. Fold Purification

 Definition: Ratio of purity after and before purification

 Calculation: Fold Purification = Final purity / Initial purity
 Dimensionless

e. Yield

 Definition: Percentage of product recovered after a step

 Calculation: Yield (%) = (Mass or Activity Recovered / Initial Mass or Activity) × 100%

f. Separation Factor

 Definition: Ratio of product/contaminant concentration ratios before and after treatment

 Calculation: α = (conc prod/conc contam)after / (conc prod/conc contam)before
 Dimensionless

9. Process Development Criteria

a. Required Purity

 Depends on application (higher for therapeutics)

 Regulatory requirements (FDA, EMA)
 Safety considerations
 Consistency requirements

b. Product Cost

 Cost of raw materials

 Number of processing steps
 Equipment and facility costs
 Scale of production
 Yield losses

c. Scalability

 Equipment availability at larger scales

 Consistent performance across scales
 Heat and mass transfer limitations
 Process control capabilities

d. Reproducibility
  Batch-to-batch consistency
 Robust process parameters
 Well-defined raw materials
 Validated analytical methods

e. Robustness

 Tolerance to variations in inputs

 Process control strategy
 Defined acceptable ranges for critical parameters
 Failure mode analysis

10. Product Specifications

 Identity (confirms it is the correct product)
 Purity (absence of process-related impurities)
 Potency (biological activity)
 Safety (absence of harmful contaminants)
 Stability (shelf-life under specified conditions)
 Physical attributes (appearance, solubility)

11. Drug Substance vs. Drug Product

Drug Substance Drug Product
Active pharmaceutical ingredient (API) Final formulated product
Bulk purified material Includes excipients, stabilizers
Intermediate in manufacturing Finished dosage form

12. Assay Attributes and Purpose

a. Precision

 Definition: Closeness of agreement between repeated measurements

 Determination: Calculate standard deviation or relative standard deviation of replicate
measurements
 Purpose: Ensures reproducible results

b. Accuracy

 Definition: Closeness of measured value to true value

 Determination: Compare to reference standards, recovery studies
 Purpose: Ensures correct quantification

c. Range
  Definition: Concentration interval with acceptable accuracy and precision
 Determination: Validate at multiple concentration levels
 Purpose: Defines useful working concentrations

d. Specificity

 Definition: Ability to measure target analyte without interference

 Determination: Test with potential interfering substances
 Purpose: Ensures measurement of only the intended analyte

e. Linearity, Limit of Detection, Limit of Quantitation

 Linearity: Linear relationship between signal and concentration

o Determined by calibration curve and regression analysis
 Limit of Detection (LOD): Lowest detectable concentration
o Determined as signal-to-noise ratio ≥ 3
 Limit of Quantitation (LOQ): Lowest concentration quantifiable with acceptable
precision
o Determined as signal-to-noise ratio ≥ 10

f. Robustness

 Definition: Capacity to remain unaffected by small variations in method parameters

 Determination: Deliberate small changes to method conditions
 Purpose: Ensures reliability during normal use

13. Analysis Methods

a. Analysis Techniques

i. Biological Activity Analysis

Method Principle Advantages Disadvantages

Animal model Test in Most thorough, best Long/slow, poor precision
animals evidence of side effects & reproducibility, resource
(administer) intensive
Cell-line Measures Reflects in-vivo Can be imprecise b/c of
derived biological activity, faster, less variances of living cells
bioassays response in expensive, less waste
cells
In-vitro Measures Simple, fast, sensitive, Overly simplified (used
biochemical interaction accurate when mechanisms and in-
assay with target vivo activity is known well)
 ii. Protein Assays

General considerations: type of materials (solids = Kjeldhal) & compatibility w/

sample, buffer, conc., proteins, (no interference).

Method Principle Considerations

Kjeldahl Used for solids/raw materials; not
Total nitrogen converted to protein
Assay specific to proteins
Bradford Blue dye (Coomassie) binds to basic Quick, sensitive; interferes with
Assay amino acids some buffers and detergents
Cu²⁺ reduced to Cu⁺ by peptide bonds, Compatible with detergents;
BCA Assay
forms complex with bicinchoninic acid interferes with reducing agents
Lowry Reaction between Cu²⁺ in alkaline Sensitive; many interfering
Assay tartrate and peptide bonds substances

iii. Gel Electrophoresis

Type Description Application

Uses reducing agents (β-mercaptoethanol, Separates subunits, analyzes
Reducing
DTT) to break disulfide bonds individual chains
Analyzes intact protein
Non-reducing No reducing agents; disulfide bonds intact
structure
No SDS, proteins retain folded structure Maintains activity, separates
Native
and native charge based on charge and size
Isoelectric Separates based on isoelectric point in pH High resolution for charge
Focusing gradient variants
2-D Isoelectric focusing followed by size sep Characterization of sample

iv. Western Blot

 Combines gel electrophoresis with antibody detection

 Identifies specific proteins in complex mixtures
 Provides information on size and approximate quantity

v. ELISA (Enzyme-Linked Immunosorbent Assay)

 Uses antibodies conjugated to enzymes for detection (bind antibody to plate)

 Highly sensitive and specific
 Quantitative or qualitative detection of proteins (low concentrations)

vi. HPLC (High-Performance Liquid Chromatography)

 Separates components based on different properties

  Types: size exclusion, ion exchange, reversed-phase, etc.
 Quantitative, reproducible, high resolution, only analytical

vii. Absorbance Measurements

 Protein absorbance at 280 nm (due to aromatic amino acids)

 Peptide absorbance at 192 nm (peptide bonds)
 Beer's Law: A = εcl
o A = absorbance
o ε = molar absorptivity (extinction coefficient)
o c = concentration
o l = path length
 Extinction Coefficient Calculation:
o ε = A / (c × l)
o For proteins: can estimate from amino acid composition
 Concentration Calculation:
o c = A / (ε × l)
o For diluted samples: c (original) = c (measured) × dilution factor

b. Endotoxin Evaluation

 Endotoxin: Lipopolysaccharides from gram-negative bacteria

 Importance: Can cause fever, inflammation, shock when injected
 Detection Methods:
o LAL (Limulus Amebocyte Lysate) assay
o Rabbit pyrogen test (animal-based)
o In vitro biochemical assays using synthetic substrates

c. Microbiological Assays

 Sterility Tests: Verify absence of viable organisms

 Bioburden Tests: Quantify microbial contamination level
 Importance: Ensures product safety and quality
 What They Measure: Presence/absence or quantity of microorganisms

d. Purity Evaluation

 Three Ways to Define Purity:

1. Mass-based: Mass of product / total mass
2. Activity-based: Activity of product / total activity
3. Relative purity: Ratio of specific contaminant to product
 Method Selection Based on Definition:

o Numerator (product quantification): HPLC, specific assays, electrophoresis

o Denominator (total mass/activity): Total protein assays, total activity assays
e. Beer's Law for Protein Concentration

 Formula: A = εcl
 Pure Protein Concentration: c = A / (ε × l)
 Extinction Coefficient: Specific to each protein
 Typical Values:
o For proteins at 280 nm: 0.5-1.5 L/g·cm
o For IgG: approximately 1.4 L/g·cm at 280 nm

14. Cell Lysis

a. Cell Structure Differences

Prokaryotes vs. Eukaryotes

 Prokaryotes: 0.1-10 μm, no nuclear membrane, few/simple organelles, often have cell
wall
 Eukaryotes: 10-100 μm, nuclear membrane present, many/complex organelles, cell wall
usually absent (except plants)

Bacteria Types

 Gram-negative: Thin peptidoglycan layer, outer membrane; easier to lyse

 Gram-positive: Thick peptidoglycan layer, no outer membrane; more difficult to lyse

b. Selection of Cell Lysis Method

Gram-negative bacteria

 Recommended Methods: Osmotic shock, detergents, moderate mechanical methods

 Considerations: Relatively easy to disrupt

Gram-positive bacteria

 Recommended Methods: Stronger mechanical methods, enzymatic (lysozyme)

 Considerations: More force required

Yeast/Fungi

 Recommended Methods: Mechanical disruption, enzymatic

 Considerations: Tough cell walls

Mammalian cells

 Recommended Methods: Gentle methods (osmotic shock, detergents)

  Considerations: No cell wall, easily damaged

c. Osmotic Pressure Calculation

 Transmembrane Pressure: ΔP = RT(Ci - Co)

o R = gas constant
o T = temperature
o Ci = internal osmolarity
o Co = external osmolarity
 Cell Breaking Condition: ΔP > cell wall mechanical strength

d. Osmolarity Calculation

 Osmolarity = Σ(concentration × number of ions)

 Example: 1M NaCl = 2 osmolar (Na⁺ and Cl⁻)

e. Cell Lysis Methods

Chemical Methods

Method Mechanism Considerations

Gentle, may interfere with
Detergents Solubilize membrane lipids
purification
Osmotic
Water influx causes cell rupture Gentle, incomplete lysis
shock
Specific, expensive, adds
Enzymatic Specific degradation of cell wall
contaminants
Extr. Proteins, remove lipids and Denature protein, special
Solvents
nucleic acids handling

Mechanical Methods

Method Mechanism Considerations

Shear forces through
Sonication Efficient, scalable, heat generation
valve
Homogenizatio Cavitation from Effective for tough, high mech stress =
n ultrasound denaturation
Bead milling Collision with beads Good for tough cells, heat, specialized equip

f. Valve-type Homogenizer Relationships

 Disruption Equation: ln(1-R) = -KNPⁿ

o R = fraction disrupted
 o N = number of passes
o P = pressure
o K, n = constants
 Temperature Impact: Higher temperatures generally increase disruption efficiency but
may damage product
 Number of Passes Impact:
o More passes → more complete disruption
o More passes → smaller debris particles
o Optimal range: typically 2-5 passes (balances disruption vs. fine debris
generation)

15. Flocculation
a. Purpose and Basic Principles

 Purpose: Aggregate small particles into larger flocs for easier separation
 Principles:
o Balance between attractive and repulsive forces
o Attractive: van der Waals, hydrophobic interactions
o Repulsive: electrostatic repulsion, hydration

b. Electrical Double Layer

i. Structure

 Stern Layer: Tightly bound counter-ions at particle surface

 Diffuse Layer: More loosely associated counter-ions
 Hydration Layer: Water molecules oriented by surface charge
 Zeta Potential: Electric potential at the slipping plane (boundary of mobile fluid)

ii. Importance

 Determines stability of colloidal suspensions

 Affects particle-particle interactions
 Influences separation efficiency

iii. Impact on Downstream Processing

 Stable colloids (high zeta potential) resist separation

 Neutralization of surface charge facilitates aggregation
 Flocculation reduces fine particles that clog filters

iv. Requirements for Flocculation

 Reduction of repulsive forces (charge neutralization)

  Addition of bridging polymers or multivalent ions
 Optimal mixing conditions

v. Thickness Factors

 Debye Radius: Characteristic thickness of double layer

 Secondary Minimum: Distance where weak attraction occurs
 Formula: κ⁻¹ = √(εₒεᵣRT/2F²I)
o κ⁻¹ = Debye radius
o εₒεᵣ = dielectric constant
o I = ionic strength
o F = Faraday constant

vi. Factors Affecting Debye Radius

 Temperature: Higher temperature → larger radius

 Ion Concentration: Higher concentration → smaller radius
 Ion Charge: Higher charge → smaller radius

c. DLVO Theory

 Critical Flocculation Concentration (CFC): Minimum concentration of flocculant

Calculation: CFC ∝ 1/z⁶ (z = valence of counter-ion)

needed

 Temperature Impact: Higher temperature requires higher CFC
 Counterion Charge Impact: Higher charge dramatically reduces CFC

d. Flocculants

 Schultze-Hardy Rule: CFC ∝ 1/z⁶

 For example:
o If monovalent ion CFC = C
o Divalent ion CFC ≈ C/64
o Trivalent ion CFC ≈ C/729