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A toolbox to reach your purification goals

GLP-1 agonists are a family of peptides that have become popular in the early treatment of diabetes type ll. Achieving the right purity for these peptides is however quite challenging.

A set of purification methods have been developed using Kromasil® stationary phases for removal of impurities in peptide crudes that can be combined in a systematic way to improve purity from crude to 99.5%. This set is called the GLP-1 agonist purification toolbox.

With a set of well adapted purification methods and routines in place, high efficiency and long column lifetime can be maintained to maximize throughput and productivity.

Liraglutide and Semaglutide

Liraglutide and Semaglutide are a couple of the main GLP-1 agonists commercially produced on the market. They have a couple of amino acid substitutions compared to human GLP-1 to slow down peptidase degradation and are both acetylated with a hydrocarbon side chain.

However, if inadequately handled during purification, they easily degrade or aggregate. Most of these issues are avoided when using the toolbox methods.

structure representation of Liraglutide
Structure representation of Liraglutide
(pdb id 4apd) using Mol* Viewer (D. Sehnal et al (2021)
Nucleic Acids Research. doi: 10.1093/nar/gkab314) and RCSB PDB.

Reaching for 99.5% purity

Crudes of Liraglutide are typically anything in the range from 10 to 50% in purity when entering downstream processing. The goal of the toolbox is to offer purification methods that will bring purity up to 99.5%.

chromatograms of various Liraglutide crudes
Typical crude purity profiles for Liraglutide

Tools selection

Operating the major factors affecting chromatography efficiency in HPLC, many different combinations of the following properties were screened:

  • Kromasil® stationayy phase
  • Mobile phase organic solvent
  • Mobile phase buffer

The screening compilation resulted in set of methods combinations with focus on the relative location of the impurities removed from the crude: back or front. The most efficient combinations have been compiled in the tables below.

Toolbox methodology

The methodical approach is to select two methods from the toolbox (tables on next page) that will target front and back impurities from the crude, respectively. In some cases this can be achieved in a single step or on a single phase.

chromatograms showing back and fron impurity groups
In a typical crude, impurities can be grouped as front and back impurities.

Toolbox combinations

Purification of Semaglutide

table with proposed combinations of phases, solvent and buffer

Purification of Liraglutide

table with proposed combinations of phases, solvent and buffer

Examples with Semaglutide

Example 1

In this example, starting with a 30% purity crude of Semaglutide, the two-steps method uses Kromasil C4 in both steps, but at different pH, obtaining 92.2% purity after the first step and then 99.5% after the second step.

chromatogram of the Semaglutide starting crude
Starting crude with focus around the product peak. Other impurities are present further away, before and after the focus area. purity: 30%

Example 1 - Purification step 1

chromatogram of the Semaglutide first prep injection
Prep injection of Semaglutide crude on Kromasil 100-10-C4
  • Conditions
  • Stationary phase: Kromasil 100-10-C4
  • Mobile phase solvent: acetonitrile
  • Mobile phase salt: trifluoroacetic acid (TFA)
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chromatograms of the Semaglutide starting crude compared to pooled analysis from separation step 1
Semaglutide starting crude compared to pooled analysis of collected fractions from separation step 1, 92.2% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid

Example 1 - Purification step 2

chromatogram of the Semaglutide second prep injection
Prep injection of Semaglutide fractions from step 1 on Kromasil 100-10-C4
  • Conditions
  • Stationary phase: Kromasil 100-10-C4
  • Mobile phase solvent: acetonitrile
  • Mobile phase salt: ammonium acetate
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chromatograms of the Semaglutide starting crude compared to pooled analysis from separation step 2
Semaglutide starting crude compared to pooled analysis of collected fractions from separation step 2, > 99.5% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid

Example 2

Example with Semaglutide using phosphate buffers at different pH on the same C4 packing media. Also starting with the 30% purity crude, the first steps at low pH reaches a 90.5% purity. The second step is run at neutral pH achieving the final 99.5% purity.

chromatogram of the Semaglutide starting crude
Starting crude with focus around the product peak. Other impurities are present further away, before and after the focus area. 30% purity.

Example 2 - Purification step 1

chromatogram of the Semaglutide first prep injection
Prep injection of Semaglutide crude on Kromasil 100-10-C4
  • Conditions
  • Stationary phase: Kromasil 100-10-C4
  • Mobile phase solvent: acetonitrile
  • Mobile phase salt: potassium phosphate, low pH
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chromatograms of the Semaglutide starting crude compared to pooled analysis from separation step 1
Semaglutide starting crude compared to pooled analysis of collected fractions from separation step 1, 90.5% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid

Example 2 - Purification step 2

chromatogram of the Semaglutide second prep injection
Prep injection of Semaglutide fractions from step 1 on Kromasil 100-10-C4
  • Conditions
  • Stationary phase: Kromasil 100-10-C4
  • Mobile phase solvent: acetonitrile
  • Mobile phase salt: potassium phosphate, neutral pH
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chromatograms of the Semaglutide starting crude compared to pooled analysis from separation step 2
Semaglutide starting crude compared to pooled analysis of collected fractions from separation step 2, > 99.5% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid

Example 3

Example using a dedicated development phase for GLP-1 receptor agonists and Kromasil Phenyl with 2 fairly neutral buffer steps with potassium phosphate and ammonium acetate, respectively.

chromatogram of the Semaglutide starting crude
Starting crude with focus around the product peak. Other impurities are present further away, before and after the focus area. 30% purity.

Example 3 - Purification step 1

chromatogram of the Semaglutide first prep injection
Prep injection of Semaglutide crude on GLPP*
*GLPP: GLP-1 Prototype phase in development
  • Conditions
  • Stationary phase: GLPP*
  • Mobile phase solvent: acetonitrile
  • Mobile phase salt: potassium phosphate, neutral pH
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chromatograms of the Semaglutide starting crude compared to pooled analysis from separation step 1
Semaglutide starting crude compared to pooled analysis of collected fractions from separation step 1, 96.6% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid

Example 3 - Purification step 2

chromatogram of the Semaglutide second prep injection
Prep injection of Semaglutide fractions from step 1 on Kromasil 100-10-Penyl
  • Conditions
  • Stationary phase: Kromasil 100-10-Phenyl
  • Mobile phase solvent: acetonitrile
  • Mobile phase salt: ammonium acetate, neutral pH
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chromatograms of the Semaglutide starting crude compared to pooled analysis from separation step 2
Semaglutide starting crude compared to pooled analysis of collected fractions from separation step 2, > 99.5% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid

Examples with liraglutide

Example 1

In this first example, starting with a 50% purity liraglutide crude, a first method using Kromasil Phenyl targets front impurities, obtaining a 94% purity. To reach a final purity of above 99.5%, a second step on Kromasil C4 is run, removing remaining back impurities.

chromatogram of the liraglutide starting crude
Starting crude with focus around the product peak. Other impurities are present further away, before and after the focus area. purity: 30%

Example 1 - Purification step 1

chromatogram of the liraglutide first prep injection
Prep injection of liraglutide crude on Kromasil 100-10-Phenyl
  • Conditions
  • Stationary phase: Kromasil 100-10-Phenyl
  • Mobile phase solvent: acetonitrile
  • Mobile phase salt: ammonium carbonate
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chromatograms of the liraglutide starting crude compared to pooled analysis from separation step 1
liraglutide starting crude compared to pooled analysis of collected fractions from separation step 1, 94% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid

Example 1 - Purification step 2

chromatogram of the liraglutide second prep injection
Prep injection of liraglutide fractions from step 1 on Kromasil 100-10-C4
  • Conditions
  • Stationary phase: Kromasil 100-10-C4
  • Mobile phase solvent: acetonitrile
  • Mobile phase salt: ammonium acetate
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chromatograms of the liraglutide starting crude compared to pooled analysis from separation step 2
liraglutide starting crude compared to pooled analysis of collected fractions from separation step 2, > 99.5% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid

Example 2

n this second example, starting with another 50% purity crude, a first method using Kromasil C8 targeting both front and back impurities, reaching a 98% purity. To achieve the final purity of above 99.5%, the second step is run on Kromasil C4, removing the remaining impurities.

chromatogram of the liraglutide starting crude
Starting crude with focus around the product peak. Other impurities are present further away, before and after the focus area. purity: 30%

Example 2 - Purification step 1

chromatogram of the liraglutide first prep injection
Prep injection of liraglutide crude on Kromasil 100-10-C8
  • Conditions
  • Stationary phase: Kromasil 100-10-C8
  • Mobile phase solvent: ethanol
  • Mobile phase salt: ammonium acetate
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chromatograms of the liraglutide starting crude compared to pooled analysis from separation step 1
liraglutide starting crude compared to pooled analysis of collected fractions from separation step 1, 94% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid

Example 2 - Purification step 2

chromatogram of the liraglutide second prep injection
Prep injection of liraglutide fractions from step 1 on Kromasil 100-10-C4
  • Conditions
  • Stationary phase: Kromasil 100-10-C4
  • Mobile phase solvent: acetonitrile
  • Mobile phase salt: ammonium acetate
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chromatograms of the liraglutide starting crude compared to pooled analysis from separation step 2
liraglutide starting crude compared to pooled analysis of collected fractions from separation step 2, > 99.5% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid

Doing it right

While peptide crudes can be challenging when repeatedly injected on an HPLC column, having a regeneration routine can be beneficial to maintain a good column lifetime and efficiency and thereby process productivity.

Troubleshooting back-pressure increase

When purifying demanding peptide crudes like Liraglutide, the column quickly gets fouled by absorbed impurities with repeated injections, even when performing a traditional 2 column volume wash between injections.

We propose here to perform an additional wash under mild conditions after a sequence of injections to quickly and fully recover column capacity and efficiency, optimizing productivity and throughput.

graph of back-pressure increase with injections
Figure showing the back-pressure over the column increasing between 30 consecutive crude injections from A to B. C shows restored normal back-pressure after the additional column wash. Chromatograms illustrating the status at these three points are shown in the following figures.

A Initial injection

chromatogram of an initial liraglutide prep injection
  • Conditions
  • Stationary phase: Kromasil 100-10-C4
  • Mobile phase solvent: acetonitrile
  • Mobile phase salt: ammonium acetate
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chromatograms pool analysis
Pool analysis, 95% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid

B After 30 sequential prep injections

chromatogram of liraglutide prep injection number 30
  • Conditions
  • Stationary phase: Kromasil 100-10-C4
  • Mobile phase solvent: acetonitrile
  • Mobile phase salt: ammonium acetate
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chromatograms pool analysis
Pool analysis, 77% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid
After 30 preparative injections, column efficiency is impaired resulting in lower purity in extracted fractions. In the preparative chromatogram, the initial peak shape is shown in light blue as reference.

C After mild regeneration wash

chromatogram of the liraglutide prep injection after mild wash
  • Conditions
  • Stationary phase: Kromasil 100-10-C4
  • Mobile phase solvent: acetonitrile
  • Mobile phase salt: ammonium acetate
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chromatograms pool analysis
Pool analysis, 95% purity
  • Conditions
  • Phase: Kromasil 100-3.5-C4 
  • Eluent: acetonitrile / water
  • Eluent salt: trifluoroacetic acid
After the regeneration wash, peak shape and thereby efficiency and purity has been practically restored. The mild wash allows for a quick turn around to proceed with new injections. In the preparative chromatogram, the initial peak shape is shown in light blue as reference.

Contact us

Are you also working on some GLP-1 agonist peptides and is interested in how this toolbox can reduce your development time and boost your productivity? Don’t hesitate to contact us and reach out to our team of skilled application scientists for more details:

Contact us

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