Investigation of Size Exclusion Columns for analysis of peptide and protein pharmaceutical

Abstract

Size Exclusion Chromatography (SEC) is a crucial analytical tool employed by Novo Nordisk for the
assessment of potential aggregation in active pharmaceutical ingredients (API) derived from peptides
and protein biopharmaceuticals. This investigation focuses on SEC analyses conducted under
denaturing conditions, utilizing Ultra High-Performance Liquid Chromatography (HULK) adapted
columns. Intriguingly, discrepancies in performance have been consistently observed over time, not
only between batches of columns from the same brand but also among different brands. Despite its
critical importance, the underlying reasons for these performance variations remain enigmatic,
necessitating a comprehensive investigation into this phenomenon.
The aim focuses on trying to answer if there is one generic method that can be developed to work for a
variety of peptides and proteins with enough suppressed secondary interactions.
Existing commercial SEC protein/peptide standards, while widely employed in industry practices,
have demonstrated limited efficacy in predicting the performance of denaturing SEC for peptides.
Through a series of controlled experiments this investigation aims to shed light upon the area,
contributing to a deeper understanding of SEC behavior in the context of peptides and protein
biopharmaceuticals.
Primarily the secondary interaction behavior is presented in increased and decreased % HMWP (high
molecular weight protein aggregation) in relation to the monomer peak but also with the elution time
shifts and tailing peaks. The separation factors between the HMWP peak and the monomer peak is
used to confirm the secondary interaction being present.
Local maxima are found for ionic suppressing additives as well as for organic modifiers. These
represent template methods that can be used when analyzing other small peptides within the same
range of size.
Conclusions:
1. There is not one type of secondary interaction for a certain type of peptide/protein in Size-
exclusion chromatography. All peptide/proteins contain a mix of functional groups, secondary
structures and more. This makes chromatographic separations of them easier in some cases
and less predictable in others.
2. There is not one generic method that works for all peptide/proteins of a certain size range.
You can create a method that will work for several proteins and peptides but now and then
you face a new peptide which requires some method development.
3. There are interparametrical relationships in Size-exclusion chromatography. If you vary one
parameter, the others will be affected as well.

Popular Abstract

Mastering Drug Development: Decoding Protein
Behavior for Safer Therapies
In a pursuit to ensure drug safety, researchers at Novo Nordisk continuously try to
understand the intricate world of proteins and peptides. The focus was on understanding
the formation of protein aggregation (both covalent and non-covalent), a phenomenon that
can impact the effectiveness of therapeutic drugs.
Using Size Exclusion Chromatography (SEC), a powerful analytical tool, the team aimed to
answer key questions:
1. Are there hidden interactions between proteins?
2. Can we predict and prevent these interactions?
3. What conditions optimize protein analysis?
The investigation revealed challenges in the reliability of traditional industry methods, with
even batches of SEC columns behaving differently. However, the team has improved the
effective strategies, by using ionic additives and specific organic modifiers, to improve the
accuracy of protein analysis. They identified template methods applicable to similar-sized
peptides.
In essence, this work is about deciphering the complexities of peptide behavior. The
researchers strive to ensure therapeutic drugs remain effective by identifying protein
aggregation and how to ultimately hinder the formation of aggregates in drugs. The goal is
to develop a universal method for analyzing proteins and peptides, advancing the safety and
reliability of drug development.
So, the next time you take medication, appreciate the unseen efforts in the lab, where
scientists work to optimize protein analysis and contribute to safer and more effective
therapies.

• author: Biuk, Hasiar
• supervisor: Margareta Sandahl; Herje Schagerlöf; Annika Sjölander
• organization: Centre for Analysis and Synthesis
• course: KASM01 20232
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Size Exclusion chromatography, secondary interactions, denaturing conditions, UHPLC, peptide analysis, analytical chemistry
• language: English
• id: 9148856
• date added to LUP: 2024-02-22 08:39:36
• date last changed: 2024-02-22 08:39:36