SCHOOL OF CHEMICAL ENGINEERING

Vellore Institute of Technology, Vellore, Tamil Nadu, India - 632014

DIGITAL ASSIGNMENT I - BIOCHEMICAL ENGINEERING (BCHE315L)

TOPIC:
Applications of Chromatographic Techniques in Bioprocesses

Team Members:
Sowmyan Nagaraj - 21BCM0094 Faculty: Dr. Sivagami K
Sathappan S - 21BCM0095 Slot: D1+TD1
Omkar Dake - 21BCM0097 Class ID: VL2024250103417
Amithese Suganth - 21BCM0099
Rohan Sebastian - 21BCM0104

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 INTRODUCTION
Chromatography is a laboratory technique for separating mixtures. It relies on differential
affinities between the components of a mixture and the stationary and mobile phases.

Phases in Chromatography:
Stationary Phase: The medium that does not move (e.g., a column filled with particles).
Mobile Phase: The medium that moves through the stationary phase (e.g., liquid or gas).

There are three types of Chromatography Based on Separation Mechanism, which

this presentation will discuss:
Affinity Chromatography: Based on specific binding interactions.
Ion-Exchange Chromatography: Based on charge differences.
Gel-Filtration Chromatography: Based on molecular size.

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 AFFINITY CHROMATOGRAPHY
Basic Principle: Molecules are separated based on specific binding interactions between the
target molecule and a ligand attached to the stationary phase. The target molecule in the mobile
phase binds to the ligand in the stationary phase, while non-target molecules are washed away.
Later, an elution step releases the target molecule by changing physical conditions such as pH or
ionic strength.

Example Ligands: Antibodies, antigens, enzyme substrates.

Applications: Primarily used for purifying proteins, enzymes, and antibodies due to its high
specificity.

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 AFFINITY CHROMATOGRAPHY
ADVANTAGES AND DISADVANTAGES OF AFFINITY CHROMATOGRAPHY:
Advantages:
High Specificity: Only the target molecule binds, leading to high purification.
Efficient: Reduces the need for additional purification steps.
Cost-Effective: Once established, it’s efficient for repeated use.

Disadvantages:
Cost of Ligands: Specific ligands can be expensive to synthesize or procure.
Limited Applications: Only works when specific binding interactions are known.
Potential Contamination: Some ligands might retain other molecules non-specifically.

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 AFFINITY CHROMATOGRAPHY
APPLICATIONS OF AFFINITY CHROMATOGRAPHY:
Protein Purification: Ion-exchange chromatography is widely used to purify proteins based on their isoelectric points
(pI), where proteins are separated by the differences in their net charge at a specific pH. Eg: The separation of serum
albumin from a mixture of blood proteins.

Nucleic Acid Purification: Ideal for separating DNA, RNA, and oligonucleotides due to their negatively charged
phosphate backbones. Eg: Isolation of plasmid DNA from bacterial lysates for research and biotechnological
applications.

Water Treatment: Utilized in removing unwanted ions (such as heavy metals) from water, making it a key technique in
water purification and desalination processes. Eg: Removal of calcium and magnesium ions in water softening.

Pharmaceutical and Food Analysis: Employed to separate and analyze charged compounds in drug formulations,
vitamins, amino acids, and food additives. Eg: Quality control in drug manufacturing to ensure purity and correct
concentration of active ingredients.

Environmental Testing: Used to analyze ions in environmental samples, such as soil and water, to monitor pollutants
and nutrients. Eg: Determination of nitrate and phosphate levels in river water for environmental impact
assessments.

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 AFFINITY CHROMATOGRAPHY
CASE STUDY: Application in Therapeutic Practices
Monoclonal antibodies (mAbs) are used extensively as therapeutics for diseases such as cancer, autoimmune disorders,
and infectious diseases. mAbs must be highly pure to ensure efficacy and patient safety. Production typically involves
culturing genetically engineered cells (e.g., CHO cells), which secrete mAbs into the culture medium, followed by a series
of purification steps. Protein A affinity chromatography is a widely used technique for mAb purification due to the high
affinity of Protein A (a bacterial cell wall protein) for the Fc (Fragment crystallizable) region of IgG (Immunoglobulin G)
antibodies. This affinity allows for selective binding of the mAb while impurities, like host cell proteins and DNA, are
washed away. The steps for the same are given below:

Sample Loading: The culture supernatant containing mAbs is loaded onto a Protein A column. Protein A, immobilized on the
chromatography resin, binds selectively to the mAbs.

Binding and Washing: As the mAb binds tightly to Protein A, other impurities (e.g., host cell proteins, DNA, and cell debris)
are washed away using a buffer that maintains binding between mAbs and Protein A.

Elution: A low-pH buffer (e.g., pH 3-4) is used to disrupt the interaction between Protein A and the mAbs, eluting the mAbs
from the column. Immediately after elution, the pH is neutralized to prevent mAb denaturation.

Polishing Steps: To ensure further purity, the mAb solution undergoes additional purification steps, like ion-exchange
chromatography or size-exclusion chromatography, to remove any remaining contaminants and aggregates.

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 GEL FILTRATION CHROMATOGRAPHY
Basic Principle: Molecule Separation is based on their size. Larger molecules elute first as they do
not enter the pores in the stationary phase, while smaller molecules penetrate the pores and elute
later. The stationary phase is made up of porous beads, which allow smaller molecules to diffuse
inside and slow down, while larger molecules pass through the void spaces.

Applications: Ideal for separating macromolecules such as proteins and nucleic acids.

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 GEL FILTRATION CHROMATOGRAPHY
ADVANTAGES AND DISADVANTAGES OF GEL FILTRATION CHROMATOGRAPHY:
Advantages:
Non-Destructive: Molecules are not altered or degraded during separation.
Simple: Easy to perform without requiring complex conditions.
Useful for Molecular Weight Determination: Helps estimate the molecular weight of proteins and
other biomolecules.

Disadvantages:
Low Capacity: Limited sample load per run.
Limited Resolution: Difficult to separate molecules of similar size.
Not Suitable for Charged Molecules: Can only separate based on size.

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 GEL FILTRATION CHROMATOGRAPHY
APPLICATIONS OF GEL FILTRATION CHROMATOGRAPHY:
Protein Purification: Used for size-based separation of proteins and biomolecules while
preserving their structure. Example: Separating immunoglobulins from other proteins based on
size.
Desalting and Buffer Exchange: Effective for removing salts or changing the buffer in a sample
without affecting the main components. Example: Desalting protein samples to prepare them
for further analysis.
Molecular Weight Determination: Estimates molecular weight by comparing elution volume
with known molecular weight standards. Example: Determining the size of unknown proteins in
research.
Removal of Aggregates: Separates monomers from aggregates to ensure product purity,
especially in pharmaceuticals. Example: Purification of monoclonal antibodies by removing
unwanted aggregates.

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 GEL FILTRATION CHROMATOGRAPHY
CASE STUDY: Desalting of Enzyme Solutions for Biochemical Analysis
Enzymes are often purified in solutions containing high concentrations of salts or other small molecules, which are used to
maintain protein stability. However, these salts can interfere with downstream biochemical assays and analysis, so desalting
is necessary before further processing or testing. Gel-filtration chromatography allows for the removal of small molecules
like salts from larger ones like enzymes without altering the enzyme’s structure or activity. There are a few major steps
required to achieve this:
Sample Loading: The enzyme solution, containing salts or other small molecules, is loaded onto a gel-filtration column
packed with beads with defined pore sizes.

Separation: As the solution moves through the column, smaller salt molecules enter the pores in the beads, while the larger
enzyme molecules are excluded from the pores and pass around them, moving faster through the column.

Elution: The enzyme elutes from the column first, followed by the smaller salt molecules. This process effectively separates
the enzyme from the salts.

Buffer Exchange: If a specific buffer is needed, the elution buffer can be chosen to replace the existing buffer in the enzyme
solution.

This technique is useful for desalting and preparing samples for sensitive arrays, particularly in spectrophotometry.

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 ANION EXCHANGE CHROMATOGRAPHY
Basic Principle: The stationary phase is positively charged, attracting and binding negatively
charged molecules (anions) in the sample. Elution is achieved by gradually increasing the ionic
strength or adjusting the pH to displace anions.

Applications: Effective for separating acidic biomolecules and nucleic acids.

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 ANION EXCHANGE CHROMATOGRAPHY
ADVANTAGES AND DISADVANTAGES OF ANION EXCHANGE CHROMATOGRAPHY:

Advantages:
High specificity for negatively charged molecules.
Can handle large sample volumes.

Disadvantages:
Requires careful pH control.
Limited to negatively charged molecules.

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 ANION EXCHANGE CHROMATOGRAPHY
APPLICATIONS OF ANION EXCHANGE CHROMATOGRAPHY:
DNA/RNA Purification: Anion-exchange chromatography is ideal for purifying nucleic acids due to
the negative charge on the phosphate backbone of DNA and RNA. Example: Isolation of plasmid
DNA from bacterial cultures for genetic engineering or molecular biology research.

Isolation of Acidic Proteins:

Commonly used for separating acidic proteins, which have a net negative charge at physiological
pH levels, by binding them to positively charged resin. Example: Purification of enzymes and
structural proteins with acidic properties for biochemical analysis.

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 ANION EXCHANGE CHROMATOGRAPHY
CASE STUDY: Purification of Plasmid DNA for Gene Therapy
Plasmid DNA is widely used in gene therapy, where it serves as a vector to deliver therapeutic genes to patients. The plasmid
DNA must be free from impurities, including RNA, proteins, and endotoxins, which can cause immune responses and reduce
the effectiveness of the therapy. Anion-exchange chromatography leverages the negative charge of DNA’s phosphate
backbone. Positively charged anion-exchange resins bind negatively charged DNA, allowing selective purification.
Lysis and Sample Preparation: Bacterial cells are lysed to release plasmid DNA, along with other cellular components like
proteins and RNA. The lysate is then clarified to remove cell debris.

Column Binding: The clarified lysate is applied to an anion-exchange column. The plasmid DNA binds to the positively
charged resin, while other impurities, such as RNA and proteins, are washed away.

Washing: The column is washed with a low-salt buffer to remove loosely bound impurities.

Elution: A high-salt buffer is used to elute the plasmid DNA from the resin. The high salt concentration disrupts the ionic
interactions, releasing the DNA from the column.

Endotoxin Removal: Additional steps, such as endotoxin removal filters or secondary chromatography, are applied to ensure
that the plasmid DNA meets the purity required for clinical use.

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 CATION EXCHANGE CHROMATOGRAPHY
Basic Principle: The stationary phase is negatively charged, attracting and binding positively
charged molecules (cations) in the sample. Elution occurs by gradually increasing the ionic
strength or changing the pH to release the cations.

Applications: Commonly used for separating basic proteins, peptides, and other positively
charged biomolecules.

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 CATION EXCHANGE CHROMATOGRAPHY
ADVANTAGES AND DISADVANTAGES OF CATION EXCHANGE CHROMATOGRAPHY
Advantages:
High Resolution: Excellent for separating proteins and peptides with similar sizes but different
charges.
Versatile: Can be used with a range of biological samples.

Disadvantages:
Sensitive to Buffer Conditions: Changes in pH or ionic strength can affect separation.
Requires Careful Optimization: Binding and elution conditions need to be optimized for each
sample type

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 CATION EXCHANGE CHROMATOGRAPHY
APPLICATIONS OF CATION EXCHANGE CHROMATOGRAPHY:
Protein Purification: Separates basic proteins and peptides for high-purity applications in
biotechnology and pharmaceuticals.

Pharmaceutical Analysis: Assists in quality control by separating bioactive components based on

charge, ensuring product efficacy and safety.

Environmental Testing: Analyzes cationic contaminants in water, such as heavy metals, supporting
environmental monitoring efforts.

Example: Separation of histones from a mixture of cellular proteins based on their positive charge,
facilitating studies on gene regulation.

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 CATION EXCHANGE CHROMATOGRAPHY
CASE STUDY: Purification of Recombinant Insulin
Cation-exchange chromatography is essential in insulin manufacturing, to provide millions of diabetic patients worldwide
with safe and effective insulin therapy. The raw insulin product needs extensive purification to meet pharmaceutical
standards, as impurities can affect efficacy and safety. Cation-exchange chromatography is often used for insulin
purification because insulin is positively charged at a slightly acidic pH, enabling it to bind to negatively charged cation-
exchange resins. The basic flow of the process is:
Sample Preparation: The crude insulin solution, which may contain host cell proteins, DNA, and other contaminants, is
prepared by adjusting the pH to around 5.5, where insulin carries a positive charge.

Column Binding: The prepared solution is loaded onto a cation-exchange column, where insulin binds to the negatively
charged resin, while other impurities either pass through or bind more weakly.

Washing: Unbound and weakly bound contaminants are washed away using a buffer, leaving only the bound insulin on the
column.

Elution: A salt gradient or pH change is applied to disrupt the interaction between insulin and the resin, allowing the pure
insulin to elute from the column
.
Polishing Steps: Following cation-exchange chromatography, the insulin undergoes additional steps, such as gel-filtration
chromatography, to remove any remaining impurities and achieve the high purity required for pharmaceutical use.

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 QUALITATIVE COMPARISON
Technique Principle Applications Advantages Limitations

Based on the charge of Protein purification, Limited to positively

Effective for separating
Cation-Exchange molecules (binding of pharmaceutical charged species; may
basic proteins and
Chromatography cations to negatively analysis, environmental require optimization for
cationic contaminants.
charged resin). testing. elution.

Based on the charge of Limited to negatively

Separation of acidic Ideal for purifying
Anion-Exchange molecules (binding of charged molecules; may
proteins and nucleic negatively charged
Chromatography anions to positively also require
acids. species.
charged resin). optimization.

Ineffective for
Separates molecules
Gentle separation with separating small
based on size; smaller Protein purification,
Gel Filtration minimal interaction; molecules from very
molecules are excluded desalting, and size
Chromatography preserves biological large ones; less
from the porous gel fractionation.
activity. resolution for closely
beads and elute later.
sized species.

Utilizes specific
Purification of specific Highly selective and Requires specific
interactions between a
Affinity proteins or efficient; can achieve ligands; may not be
ligand and target
Chromatography biomolecules with high high purity in a single applicable for all
molecules (e.g.,
specificity. step. proteins.
antibodies, enzymes).

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 REFERENCES
F, G. (2020). Cation Exchange Chromatography: Principles and Applications. Analytical Chemistry, 92(1), 123-134.
doi:10.1021/acs.analchem.9b04258
Zhang, J., & Wang, Z. (2019). Recent Advances in Anion Exchange Chromatography: Applications in Proteomics. Journal of
Chromatography A, 1581, 25-36. doi:10.1016/j.chroma.2018.12.021
Barylyuk, K., & Zubareva, A. (2018). Gel Filtration Chromatography: A Review of Current Applications. Journal of
Chromatography B, 1081-1082, 34-44. doi:10.1016/j.jchromb.2018.04.020
Hage, D. S., & McCormick, R. (2018). Affinity Chromatography: Principles and Applications. Journal of Chromatography A,
1541, 23-35. doi:10.1016/j.chroma.2017.12.031
Riemer, S., & Oh, C. (2020). High-Performance Liquid Chromatography for Protein Analysis. Biotechnology Advances, 38,
107444. doi:10.1016/j.biotechadv.2019.107444
Pabst, M., & Sinner, E. (2021). Advances in Protein Purification: An Overview of Techniques. Biotechnology Letters, 43(1),
1-13. doi:10.1007/s10529-020-02981-y
Prabhu, K. S., & Narayanaswamy, R. (2019). Ion-Exchange Chromatography for Biopharmaceutical Purification. Journal of
Pharmaceutical Sciences, 108(4), 1210-1222. doi:10.1016/j.xphs.2019.01.028
Bonifacio, C. M., & Lima, F. D. (2020). Application of Gel Filtration Chromatography in Biomolecular Analysis. Journal of
Molecular Recognition, 33(1), e2812. doi:10.1002/jmr.2812
Lestari, S. K., & Yulianto, E. (2021). The Role of Affinity Chromatography in Bioprocessing. Biochemical Engineering
Journal, 164, 107842. doi:10.1016/j.bej.2020.107842
Zhang, H., & Wang, Y. (2018). Integration of Cation Exchange and Affinity Chromatography for Protein Purification.
Molecules, 23(11), 2954. doi:10.3390/molecules23112954

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 THANK YOU

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