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Welch Solution for Biological Sample Analysis

Welch Solution for Biological Sample Analysis

Table of contents
Introduction Size Exclusion Chromatography Application Examples Reversed-phase Liquid Chromatography Application Examples Hydrophobic Interaction Chromatography Application Examples Ion Exchange Chromatography Application Example

Introduction

In chromatographic separation, biological molecules present unique challenges due to their structural complexity, wide molecular weight range, and sensitivity to environmental conditions. From therapeutic peptides and oligonucleotides to monoclonal antibodies and recombinant proteins, each class of biomolecule requires chromatographic methods tailored to its specific physicochemical properties.

Welch Materials offers a comprehensive portfolio of chromatography solutions designed to address the diverse analytical needs of biological samples. In this article, we introduce our chromatography platforms for biological sample analysis, covering size exclusion chromatography (SEC), reversed-phase liquid chromatography (RPLC), hydrophobic interaction chromatography (HIC), and ion exchange chromatography, along with their key applications in the analysis of peptides, nucleic acids, antibodies, and proteins.

Note: This article only presents some of the applications from the document. Download the full solution document at the end of the article for more application examples.

Size Exclusion Chromatography

Read more: Mitigating Non-specific Adsorption in Size Exclusion Chromatography with Xtimate Bio SEC

Xtimate Bio SEC series is Welch Materials' core SEC solution for biosamples. Its stationary phase utilizes a unique surface modification technology, prepared by bonding hydrophilic high-molecular-weight polymers onto an ultra-pure silica matrix. Controlled chemical modification ensures reliable batch-to-batch reproducibility. The hydrophilic polymer is bonded uniformly and completely, providing extensive coverage of the silica surface and minimizing non-specific adsorption (NSA) of biological samples such as proteins.

Application Examples

Analysis of monoclonal antibody aggregates

  • Column: Xtimate Bio SEC-300 (5 μm, 4.6×300 mm)
  • Mobile Phase: 50 mM phosphate + 300 mM NaCl buffer
  • Flow Rate: 0.2 mL/min
  • Detector Wavelength: 280 nm
  • Column Temperature: 25 °C
  • Injection Volume: 10 μL
Chromatogram of monoclonal antibody aggregates

Determination of recombinant type III humanized collagen content

Chromatogram of recombinant type III humanized collagen

Analysis of octreotide acetate aggregate

Reversed-phase Liquid Chromatography

Ultisil and Xtimate are Welch's two primary HPLC column series, with broad ranges of reversed-phase and normal-phase stationary phases to choose from.

The Ultisil Series uses fully porous spherical silica as a matrix, combining proprietary stationary phase bonding and silica surface treatment technologies. These columns offer high resolution, excellent peak shapes, and low protein adsorption. Common reversed-phase Ultisil bonded phases include C18, C8, C4, C3, etc.

The Xtimate Series utilizes a hybrid silica matrix, capable of withstanding harsher analytical conditions. It exhibits exceptional stability and longevity under high temperatures and extreme pH mobile phases. Its double-endcapping process minimizes residual silanol activity, ensuring optimal peak shapes.

Application Examples

Determination of thymalfasin related substances

  • Column: Ultisil LP-C18 (5 μm, 4.6×250 mm)
  • Mobile Phase: A) (NH4)2SO4 buffer / ACN = 90/10; B) (NH4)2SO4 buffer / ACN = 50/50
  • Flow Rate: 0.8 mL/min
  • Detector Wavelength: 210 nm
  • Column Temperature: 30 °C
  • Injection Volume: 20 μL
  • Sample Concentration: Main 0.5 mg/mL; Impurities 2.5 μg/mL each
  • Gradient:
    Time (min) A% B%
    0 90 10
    15 84 16
    40 83 17
    65 83 17
    100 50 50
    105 50 50
    106 90 10
    120 90 10
Chromatogram of thymalfasin related substances

Analysis of oligonucleotide

  • Column: Xtimate C18 (5 μm, 4.6×250 mm)
  • Mobile Phase: A) PB / MeOH = 95/5; B) PB / MeOH = 75/25
  • Flow Rate: 1.0 mL/min
  • Detector Wavelength: 260 nm
  • Column Temperature: 25 °C
  • Injection Volume: 10 μL
  • Gradient:
    Time (min) A% B%
    0 100 0
    20 100 0
    30 50 50
    35 0 100
    40 0 100
Chromatogram of oligonucleotide

Determination of human G-CSF purity

  • Column: Ultisil XB-C18 (5 μm, 4.6×250 mm, 300 Å)
  • Mobile Phase: A) 0.1% TFA-water; B) 0.1% TFA-ACN
  • Flow Rate: 1.0 mL/min
  • Detector Wavelength: 214 nm
  • Column Temperature: 30 °C
  • Injection Volume: 2 μL
  • Gradient:
    Time (min) A% B%
    0 50 50
    30 10 90
    31 50 50
    45 50 50
Chromatogram of human G-CSF

Determination of lactoferrin

  • Column: Xtimate C4 (3 μm, 4.6×150 mm, 300 Å)
  • Mobile Phase: A) 0.1% TFA-water; B) 0.1% TFA-ACN
  • Flow Rate: 1.0 mL/min
  • Detector Wavelength: 220 nm
  • Column Temperature: 60 °C
  • Injection Volume: 10 μL
  • Gradient:
    Time (min) A% B%
    0 85 15
    15 50 50
    15.1 5 95
    18 5 95
    18.1 85 15
    25 85 15
Chromatogram of lactoferrin

Hydrophobic Interaction Chromatography

Read more: Hydrophobic Interaction Chromatography: Your Best Teammate in the Analysis of ADCs

Advanchrom HIC series is Welch Materials' key tool in the separation of antibody-drug conjugates (ADCs) and the analysis of drug-to-antibody ratio (DAR). Advanchrom Bio HIC-Butyl is a high-performance column designed for protein separation based on hydrophobic interaction chromatography, utilizing an advanced macroporous silica matrix combined with a unique surface bonding technique. Advanchrom HIC-Butyl, on the other hand, is an ideal choice for low hydrophobicity antibodies, proteins, and ADCs.

Application Examples

DAR analysis of disitamab vedotin reference formulation

  • Column: Advanchrom Bio HIC-Butyl (4.6×100 mm, 5 μm)
  • Mobile Phase: A) 50 mM PB + 2.0 M (NH4)2SO4, pH 7.0; B) 50 mM PB, pH 7.0 / IPA = 75/25
  • Flow Rate: 1.0 mL/min
  • Detector Wavelength: 280 nm
  • Column Temperature: 30 °C
  • Injection Volume: 5 μL
  • Gradient:
    Time (min) A% B%
    0 90 10
    1 90 10
    25 0 100
    30 0 100
Chromatogram of disitamab vedotin reference formulation

Analysis of rituximab

  • Column: Advanchrom HIC-Butyl (4.6×50 mm, 3 μm)
  • Mobile Phase: A) 1.0 M CH3COONH4; B) 25 mM CH3COONH4 / ACN = 70/30
  • Flow Rate: 0.5 mL/min
  • Detector Wavelength: 280 nm
  • Column Temperature: 30 °C
  • Injection Volume: 10 μL
  • Gradient:
    Time (min) A% B%
    0 90 10
    2 90 10
    15 0 100
    20 0 100
Chromatogram of rituximab

Ion Exchange Chromatography

Advanchrom SCX is a high-performance strong cation exchange (SCX) column based on advanced monodisperse non-porous polymer microspheres and utilizes a proprietary surface bonding technology. It is specifically designed for the separation and characterization of charge variants in mAbs, bsAbs, fusion proteins, and ADCs.

Application Example

Analysis of NIST mAb charge variant

  • Column: Advanchrom SCX (4.6×150 mm, 5 μm)
  • Mobile Phase: A) 20 mM MES (pH 6.5); B) 20 mM MES (pH 6.5) + 0.5 M NaCl
  • Flow Rate: 0.3 mL/min
  • Detector Wavelength: 280 nm
  • Column Temperature: 30 °C
  • Injection Volume: 2 μL
  • Sample Concentration: 10 mg/mL
  • Gradient:
    Time (min) A% B%
    0 90 10
    30 70 30