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How to Combine Chromatography Techniques for Antibody Purification

Purity and Yield: Get the Balance Right

Lotta Hedkvist, Severine Lebarq, Axel Parbell

Antibody purification, purity and yield: get the balance right

Antibody purification protocols are typically challenged by two factors:

  •  Capturing as many antibodies as possible without degrading the sample.
  •  Removing the remaining impurities and minimizing aggregate content.

With that in mind, what should be considered when planning your antibody purification experiment?

  • The choice of chromatography techniques depends on the purity requirement of your antibody of interest. The required antibody purity level will depend on your application, as shown on the table below.
  • Before you start, carefully define your objectives and consider that in general, every added purification step will increase purity but decrease total protein recovery1 and yield2

Typical applications                                                 Purity level

Mass spectrometry, Antigen for immunization              Moderate to high, 80% to 90%

Functional studies, Structural studies                          Very high, 95% to 99%

Structural studies, Therapeutic proteins                       Highest > 99%

1 Recovery = The relative amount of target protein (%) that is retrieved after purification compared with amount loaded on the column.

2 Yield = Amount of target protein obtained after a purification step, or after the entire purification (multiple steps).

What do antibody purification protocols look like?

The picture describes typical proven technique combinations for the purification of antibodies.

All antibody purification protocols typically start with an affinity chromatography step (AC).

It enables the isolation of antibodies from initial sample (e.g., serum, cell culture)

1-step protocol and 2-step protocol:

Antibody purification, purity and yield: get the balance right

Antibody purification protocols are typically challenged by two factors:

  •  Capturing as many antibodies as possible without degrading the sample.
  •  Removing the remaining impurities and minimizing aggregate content.

With that in mind, what should be considered when planning your antibody purification experiment?

  • The choice of chromatography techniques depends on the purity requirement of your antibody of interest. The required antibody purity level will depend on your application, as shown on the table below.
  • · Before you start, carefully define your objectives and consider that in general, every added purification step will increase purity but decrease total protein recovery1 and yield2

Typical applications                                                   Purity level

Mass spectrometry, Antigen for immunization              Moderate to high, 80% to 90%

Functional studies, Structural studies                           Very high, 95% to 99%

Structural studies, Therapeutic proteins                       Highest > 99%

1 Recovery = The relative amount of target protein (%) that is retrieved after purification compared with amount loaded on the column.

2 Yield = Amount of target protein obtained after a purification step, or after the entire purification (multiple steps).

What do antibody purification protocols look like?

The picture describes typical proven technique combinations for the purification of antibodies.

All antibody purification protocols typically start with an affinity chromatography step (AC).

It enables the isolation of antibodies from initial sample (e.g., serum, cell culture)

1-step protocol and 2-step protocol:

The 1- and 2-step protocols are the recommended best choices for research use. After the AC step, the purity level is usually high (> 90%). However, if there is a need to remove antibody aggregates and/or fragments to obtain monomeric antibodies, then a size exclusion chromatography (SEC) step is recommended. The purity level after a 2-step purification will be very high (95% to 99%).

3-step protocol:

Consider using the 3-step protocol for scale-up or process development. SEC is not used as a final step to remove aggregates, fragments, or other impurities, due to the limitation of sample volume. Instead, a combination of IEX (ion exchange chromatography) steps is used.

After the AC step, cation exchange chromatography (CIEX) is first used in a "bind elute" mode to further remove host cell proteins (HCP) leached protein A ligand, mAb aggregates, fragments, and other isoforms.

Then the sample is applied to AIEX (anion exchange chromatography) column in a "flow through" mode for a final removal of remaining impurities of HCP, DNA, and viruses.

The antibody purity will then be extremely high (>99%).

Importance of buffer exchange and concentration in the antibody purification protocol

 To make the sample compatible with the following steps or experiments, it might be necessary to use a desalting column for buffer exchange, and/or a concentration unit to reduce the sample volume.

Which chromatography columns are recommended for each step?

Affinity chromatography step:

  • HiTrap™ Protein A HP or HiTrap Protein G HP
  • HiTrap MabSelect™ PrismA or HiTrap MabSelect SuRe™
  • HiScreen™ MabSelect PrismA or HiScreen MabSelect SuRe (for PD or scale-up)

 Ion exchange steps:

  • HiTrap Capto™ S ImpAct or HiScreen Capto S ImpAct (CIEX)
  • HiTrap Capto Q or HiScreen Capto Q (AIEX)

Size exclusion Step:

  • Superdex™ 200 Increase
  • HiLoad™ Superdex 200 pg
  • HiPrep™ Sephacryl™ S-300 HR

Buffer exchange is best done using HiTrap 5 mL or HiPrep 26/10 Desalting columns suitable for sample volumes up to 15 mL.

More details about how to find the best column depending on purity needs and sample source can be found in the Antibody purification and analysis brochure (29251059AC) available on gelifesciences.com.

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