Optimization of two high-performance size-exclusion chromatography methods with diode-array detection for protein quantification and purity assessment

Abstract

Background: There is a need to develop methods of determining concentration and purity for
two new affinity ligands, protein T and protein H, in order to ensure the quality of an
industrial production of said proteins.
Aims: To develop a High Performance Size Exclusion Chromatography, HPSEC, method to
determine concentration and purity of protein T and to improve an existing HPSEC method
for concentration and purity determination of protein H that show selectivity, display linearity
between peak area and injection amount and give repeatable results.
Methods: For protein T, Xbridge Premier Protein and Superdex Increase columns were used
of the lengths 15 and 30 cm together with 50mM and 0.3M Sodium phosphate buffers and
PBS and mobile phases. For protein H, only a 15 cm Superdex Increase column was used
with 0.3M Sodium phosphate buffer. Different reducing conditions during sample preparation
were tested. The linearity and measuring ranges were determined by injecting different
volumes of reference standard and evaluating . Repeatability was tested with three triplicates
of reference standards across three occasions with the same equipment and running
conditions.
Results: For protein T the 15 cm Xbridge column with 50mM Sodium Phosphate buffer was
selected. Little difference between reduction conditions with exception of reduction at high
temperature which resulted in degradation or aggregation for both proteins. Acceptable
selectivity and separation between main peak and impurities was demonstrated for both
methods. Both methods were linear and gave repeatable results in the measuring ranges 2.43 –
16.98µg, 4.85 – 16.98µg and 2.56 – 15.37µg for purity determination of protein T,
concentration determination of protein T and both purity and concentration determination
protein H, respectively.
Conclusion: Two methods, one for protein T and one for protein H, were developed which
met qualification criteria use in quality control.

Popular Abstract

An important part of pharmaceutical production is the purification of the active ingredient which can be done, in part, using proteins called affinity ligands which selectively bind specific targets. Just like the active ingredients in drugs, companies must be sure that these affinity ligands don’t contain harmful impurities and fulfil product specifications in order to protect patient safety.

A common efficient method for determining protein concentration and purity is called high performance size exclusion chromatography, HPSEC. HPSEC is a method where a solution flows through a tube filled with porous particles called a column, which separates compounds according to how fast they travel through the column. How fast something travels depends on the size of the compound. Different affinity ligands have different properties which may impact how they interact with the column which may distort results. Therefore suitable parameter conditions must be determined for each affinity ligand to be measured. The aim of this study is to develop to methods by finding suitable HPSEC parameter conditions for determining purity and concentration of two affinity ligands, here called protein T and protein H. Both methods should be separate the affinity ligands well from impurities and be linear and repeatable.

To this end, four columns were tried; two different sizes for the porous particles, and two different tube lengths. Solutions with different salt concentrations were used to pass through the column as well as different times and temperatures of preparing the samples were tried so that the ligands were in their active states. Once suitable parameters had been selected, different protein amounts were used to see for what sample concentrations the method would be linear. The concentration and purity of a sample were determined on three different days using the final selected parameters to test if the method was repeatable.

The results showed the column with smaller particles were better at separating out the affinity ligand, and worked best in combination with the solution with a low salt concentration. The longer columns could separate more compounds, but took long time for each analysis (40 min vs 20 min) so the shorter column with small particles was selected. High temperature, 85℃, seemed to degrade both affinity ligands the longer they were reduced, while little difference were seen at lower temperatures, 30 and 60℃, no matter the time. Both methods were linear and gave repeatable results when using 2.43 – 16.98µg, 4.85 – 16.98µg and 2.56 – 15.37µg of protein for purity determination of protein T, concentration determination of protein T and both purity and concentration determination protein H, respectively.

All in all, two methods, one for protein T and one for protein H, were developed that could be used for controlling the quality for later use in the pharmaceutical industry.

• author: Lane, Camilla
• supervisor: Marika Slivacka; Peter Spégel
• organization: Centre for Analysis and Synthesis
• course: KASM01 20231
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: Method qualification, Protein concentration, Protein Purity, High Performance Size exclusion chromatography, Quality control, Analytical chemistry
• language: English
• id: 9127744
• date added to LUP: 2023-06-21 09:44:09
• date last changed: 2023-06-21 09:44:09