Lecture 8:

Chromatography and
Column Packing
Learning Objectives
1. Outline the commonly used chromatographic
techniques

2. Describe affinity, ion exchange and

hydrophobic interaction chromatography

3. Discuss why different chromatographic

techniques are used in a typical bioprocess
Topics

Overview
Column Packing
Operation Cycle
Affinity
IEX
HIC
Overview
What is Purification?

Recovery and purification of biopharmaceuticals, removal of

contaminants and impurities
Typical Process Feedstream
The target molecule is usually a small percentage (<0.5%) of a
complex molecular mixture

Legend

Contaminant
Molecules

Target Protein
Main Methods of Protein Recovery
Centrifugation
Uses centrifugal force to separate based on the
principles of sedimentation

Filtration
Relies upon size difference or charge effect to
separate materials

Chromatography
There are many different types of chromatography
available:
Ion exchange
Size exclusion
Affinity
HIC
Column Chromatography
Used to purify an individual compound from a mixture
of compounds

Vertical column packed with a stationary matrix (called

stationary phase or resin or chromatography media)

Mobile solution use to carry protein through column

(called mobile phase)

A complex sample is passed through the stationary

matrix that consists of a solid support with specific
characteristics defined by the support’s make-up
and/or chemistry
Column Chromatography Phases
1. Stationary Phases 2. Mobile Phase
Stationary Phase
The stationary phase is a solid matrix retained within a column
through which the mobile phase passes

The stationary phase, or matrix, in the form of small particles,

is packed into the column

A complex sample is passed through the stationary matrix that

consists of a solid support, with specific characteristics defined
by the support’s make-up and/or chemistry
Mobile Phase
The mobile phase is the part of the chromatographic system
that carries the solutes through the stationary phase

Liquid mobile phases are used to adjust the chromatographic

separation and retention in liquid chromatography

Stationary Mobile
Phase Phase
Flow Through vs. Bind and Elute
Chromatography
Bind and Elute: Flow-through:
The molecule of interest binds to The molecule of interest does not
the chromatography bead bind to the chromatographic bead

Flow Flow
Chromatography Chromatography
bead Sample bead
Contaminant

Sample is bound Contaminant is

bound
Contaminant not
bound
Sample not
bound
Components of Chromatography System
Main components include buffer tanks (bags), pump, column, sample injector system
and the detector:
1. The buffer tanks hold the mobile phase.
2. The pump is used to generate a specified flow of the mobile phase.
3. The injector introduces the solvent into a phase stream that carries the sample into
the column.
4. The chromatography column contains the stationary phase
5. A detector is needed to see the separated compound bands as they elute from the
column. This is generally a UV detector at 280nm.

Detection System
Chromatography Column
Sample Injector System

Buffer Supply
(inline buffer dilution system)
Pump
Chromatography Skid P&ID
P&ID
Piping and Instrument Diagram
Sample
Inlet
Bubble UV
Buffer Trap Filter Sensor Column
Inlet Flow
Meter

Pressure
Sensor

Pump pH Conductivity
Sensors
Björling, T. B. and M. (2021, March 17). High-quality
chromatography system meets predesigned deltav for
integrated manufacturing. Cytiva. Retrieved June 6, 2022, from
https://www.cytivalifesciences.com/en/us/news-
center/chromatography-system-for-manufacturing-10001
Column Anatomy

Top Adjustable Plate

Acrylic
Colum Frit or Stainless Steel
Column
Bed Support Column Tube
Tube
Bottom Fixed Plate

Column Stand

Pall Resolute Pall Resolute

400mm Acrylic 1200mm Stainless Steel
Column Bed Support
Acts like a sieve and keeps stationary phase in column tube

Pores must be smaller than resin beads

Can be stainless steel or polypropylene

Example of Chromatogram

Protein of
Interest

Sample
Loading

Wash Elution
Question
What are the two phases of column chromatography?

Stationary Phase

Mobile Phase

(Post your answers in the chat – get those participation marks!)​

Poll
Protein properties determine the type of chromatography to
be used.

True

Or

False
 Protein Properties Determine
Chromatography Method
Affinity
Size Exclusion Size & Shape Properties Affinity
Chromatography (3D Structure) Density (Ligand Chromatography
Binding)

Ion Exchange/
Charge & Chromatography
Mixed Mode/
Isoelectric Solubility Mobile Phase
Hydroxypatite
Point (Buffer)
Chromatography

Ion Exchange/
Aggregation Size Exclusion
Mixed Mode/
Hydrophobicity (Reversible or Chromatography/
Hydroxypatite Irreversible) Buffer
Chromatography
Column Packing
What Is Column Packing?

Column packing is a process of putting stationary phase

(resin) into a column.

Stationary Phase
Why Is Column Packing So Critical?

For maximum chromatographic efficiency, high product yield and purity, you must
have a homogeneous packed bed every time you perform a separation.

A poorly packed column will cause uneven flow within the packed bed, resulting in:
adverse product quality
loss of product yield
Column Packing Objectives

Achieve a homogeneous bed

Uniform flow profile
High efficiency
Good symmetry

Reproducible chromatogram from batch to batch

Prepacking Factors To Consider
Before packing a column, the following must be considered:

Resin (Stationary Phase)

Particle size, resin slurry concentration , propensity of the resin to generate fines
and compression factor.

Buffer (Mobile Phase)

Chemical compatibility, shrinkage or swelling of resin in mobile phase, viscosity, pH
stability and mobile phase interaction to resins.

Column
Routine preventive maintenance , routine cleaning , temperature effect on column
hardware

Bed supports/Frits
Clean frit from particulate. Free of entrapped air
Packing Methods

1. Gravity Settling
2. Flow Pack
3. Pack in Place
4. Syringe Pack
 1. Gravity Settling
Gravity settling is the easiest approach to column packing

The resin and packing buffer are slurried and poured into the column

Slurry
poured Slurry in After 30 mins After 60 mins
in Column
Packing
Buffer Packing
Buffer

Resin
Resin

Empty Column
2. Flow Packing
Pack the column in a controlled temperature environment, ideally at the same temperature at
which it will be operated

Completely equilibrate the temperature of both the column itself and the resin before packing
No Further
Buffer Buffer Buffer Compression
Pumped In Pumped In Pumped In Occurs
Piston is
Column Lowered Piston is Piston is
Piston Lowered Lowered

Slurry
Qualifying the Column Packing
Once the column is packed, it is qualified by performing the following tests:
Column Efficiency
Asymmetry

Why Column is tested after packing?

GMP Requirement
Ensures consistency
Asymmetry Factor (As)
The asymmetry factor As describes the
deviation from an ideal Gaussian peak shape
and is calculated from the peak width at 10%
of peak height

The peak asymmetry factor should be as close

as possible to 1, and the shape of the peak
should be as symmetrical as possible.
Resin Bead
Operation Cycle
Typical Column Chromatography
Operations 1

Equilibration
Storage 7 2 Sample Loading

Regeneration 6 3 Wash

Sanitation 5 4 Elution
Column Chromatography Operations
1. Column Equilibration
To achieve a stable and equal distribution of a desired buffer, a column packed with a
chromatography medium has to be run in the respective buffer to a point where pH,
conductivity and UV, measured at the column outlet, are identical to the respective values of
the applied buffer.

Choose buffer composition & pH to maximise product binding & minimise binding of impurities,
without damaging the protein.

Equilibration Equilibration
Buffer Buffer

Column NOT Equilibrated

Equilibrated Column
Column Chromatography Operations
2. Sample Loading
The sample size will relate to the total capacity of the matrix.
The capacity of the matrix can be determined at lab scale by
passing a sample of known product through a known volume of
matrix until the sample appears in the eluent.

Sample is applied to column at same flow rate as mobile phase.

Sample must be prepared to ensure binding to the stationary

phase (the resin).

3. Wash Step Example of binding to stationary

May be same as Equilibration buffer and a different buffer to phase
remove impurities.
Column Chromatography Operations
4. Elution Methods
The target molecule is bound to the resin.
Change in pH (e.g. pH 2-3.5), change in ionic strength, etc. The column should be
re-equilibrated to neutral pH immediately.

A compromise may have to be made between the harshness of the eluent required
for elution and the risk of denaturing the eluted material or damaging the ligand on
the affinity medium.

Proteins can be eluted in a stepwise, gradient or isocratic format.

Isocratic Composition of eluent does NOT

Elution change during the purification run
Column Chromatography Operations
5. Cleaning
Chromatography is not a sterile process. Chromatography columns and Chromatography
Systems are sanitised to reduce bioburden

Chromatography columns have several cleaning challenges – difficult to achieve high flow
rates to clean the columns; inaccessibility; damage to the resin; difficult to clean O-rings, etc;
ancillary equipment such as pumps, hoses, etc. must be cleaned separately

Disassembly of column is labour intensive

Column Chromatography Operations
6. Regeneration
Returning the stationary phase in a chromatography column to its initial state after elution.
Mobile phase is passed through the column stepwise or in a gradient. The stationary phase is
solvated to its original condition.

Regeneration can also refer to bringing back any column to its original state (e.g., the removal
of impurities with a strong solvent).

Column regeneration is an important step of protein purification.

7. Storage
Stored in appropriate buffer/ storage solution to prevent microbial growth.
Affinity
Affinity Chromatography
Affinity chromatography separates molecules based on the reversible interaction
between target protein and the specific ligand attached to a chromatography matrix.

Target
Protein

Ligand
attached
Custom affinity chromatography service. (n.d.). Retrieved June 6, 2022, from
to matrix https://www.creative-biostructure.com/custom-affinity-chromatography-service-
257.htm
 Different Games Require Different Balls
Mobile Phase
Sample consisting of different molecules
applied to the column

Molecules with the affinity to the stationary

phase will bind to the resin

Molecules with no affinity to the stationary

Stationary Phase
phase will travel with mobile phase through
the column

Bead Ligand
Advantages vs. Disadvantages:
Affinity Chromatography
Advantages: Disadvantages:
High specificity Ligand availability
High selectivity High cost
High recovery Often exhibit poor stability
Purity increases of 1000-fold Ligand leakage
Ion Exchange Chromatography
(IEX)
Proteins Carry a Charge
All proteins are made up of different combinations of the 20 amino acids

Some of these amino acids possess side groups (R groups) that are either positively or
negatively charged.
Ion Exchange Chromatography(IEX):
Principles
IEX separation uses reversible interactions between charged
molecules and oppositely charged Ion-Exchange resin

Cation Exchange
Uses negatively charged resin
Binds molecules with net positive surface charges

Anion Exchange
Uses positively charged resin
Binds molecules with net negative surface charges
Advantages vs. Disadvantages:
IEX Chromatography
Advantages: Disadvantages:
Ligand availability Low specificity
Inexpensive Sample must be applied at low
High recovery ionic strengths and controlled pH
(may require a buffer exchange
steps for sample and elution)
Hydrophobic Interaction
Chromatography (HIC)
Hydrophobic Interaction
Chromatography (HIC)

Hydrophobic interaction chromatography is most commonly used for:

Polishing - to remove trace amounts of contaminants or closely
related substances such as structural variants of the target protein.
 Hydrophobic Interaction
Chromatography
The three-dimensional structure of a protein is a result of intra-molecular interactions as well
as interactions with the surrounding solvent.

In the case of readily soluble proteins, this solvent is water, and hydrophobic side chains are
therefore typically driven to the interior of the protein.

Hydrophobic
Hydrophilic Hydrophobic Residues
Residue Core Exposed

Hydrophobic Protein
Residue Water High Salt Buffer
Advantages vs. Disadvantages:
Hydrophobic Interaction Chromatography

Advantages: Disadvantages:
Samples with high ionic Low specificity
strength can be used High salt concentration in
Large sample volumes can waste
be loaded
Sample eluted with low salt
Poll
Affinity chromatography separates based on:

A. Positive charge

B. Biological function or individual chemical structure

C. Hydrophobic side chains

D. Chromatography
Topics Review

Overview
Column Packing
Operation Cycle
Affinity
IEX
HIC
Questions?
References
Air Traps. Cytiva. (n.d.). Retrieved June 6, 2022, from https://www.cytivalifesciences.com/en/us/shop/chromatography/tools-and-accessories/other-accessories/air-traps-p-
06317
Björling, T. B. and M. (2021, March 17). High-quality chromatography system meets predesigned deltav for integrated manufacturing. Cytiva. Retrieved June 6, 2022, from
https://www.cytivalifesciences.com/en/us/news-center/chromatography-system-for-manufacturing-10001
Custom affinity chromatography service. (n.d.). Retrieved June 6, 2022, from https://www.creative-biostructure.com/custom-affinity-chromatography-service-257.htm
Cytiva. (n.d.). Retrieved June 6, 2022, from https://www.cytivalifesciences.com/en/us