HPLC

Bioanalytics for Antibodies and Proteins
ApplicationsTechnology

HPLC – Multiple analytical applications

Meet our one-for-all tool for protein and antibody analytics: The Agilent 1260 Infinity HPLC system with SEC, HIC, and IEX colums, coupled to Wyatt MALS and RI detectors.

This system can unravel the all the important features of your protein or antibody sample: Molecular weight, oligomerization state, hydrophobicity, charge-state and charge-variants.

The ultrafast service from 2bind includes a detailed report and definitive answers to quality and developability questions.

Size-Exclusion Chromatography
SEC-MALS

OLIGOMERIZATION, AGGREGATION, PURITY, FRAGMENTATION


Retention time
The smaller a molecule, the longer its retention time. Gives information on molecular size/weight, as well as the oligomerization state and aggregation content of the sample.


Covalent and colloidal integrity
Protein fragments, oligomers, and aggregates are visible in SEC-MALS profiles.


Stoichiometry of protein complexes
Protein complexes are larger in size than their individual subunits. Therefore, protein complexes and their stoichiometry can be differentiated in SEC-profiles and MALS data.


Purity
Size-dependent separation of proteins shows impurities and byproducts of a sample.

 

MOLECULAR WEIGHT


MALS (Multi-Angle Light Scattering)
High-precision and calibration-free measurement of protein molecular weight via scattered light. 

 

Hydrophobic-Interaction Chromatography
HIC

HYDROPHOBICITY


Retention time
The longer the retention time of a sample on a HIC-column, the higher its hydrophobicity. Important developability marker for antibodies.


Hydrophobicity of antibodies
Determined in relation to established controls. Important developability marker.


Purity
Hydrophobicity-based, size-independent separation of proteins shows impurities and byproducts of a sample from a different perspective than SEC-MALS.

 

MOLECULAR WEIGHT


MALS (Multi-Angle Light Scattering)
High-precision and calibration-free measurement of protein molecular weight via scattered light.

Ion-Exchange Chromatography
IEX

CHARGE VARIANTS


Retention time
Defined by the charge of the molecule, the column matrix, and the ionic strength of the mobile phase. Gives information on the charge state of a protein and possible charge variants. Important developability marker for antibodies.


Charge variants of antibodies
Determined in relation to established controls. Important developability marker.


Post-translational modifications
PTMs can influence the surface charge distribution of proteins and can therefore be identified and differentiated from ion-exchange profiles.


Purity
Charge-based, size-independent separation of proteins shows impurities and byproducts of a sample from a different perspective than SEC-MALS.

 

MOLECULAR WEIGHT


MALS (Multi-Angle Light Scattering)
High-precision and calibration-free measurement of protein molecular weight via scattered light. 

HPLC is a universal tool for protein and antibody characterization

High performance liquid chromatography (HPLC) separates the contents of liquid or solid samples on chromatography columns. The type of column and the running buffer define the parameterd of separation, the application of the system, and the questions that can be answered with it.

Antibodies

SEC-MALS is a crucial assay for antibody quality control. A clean SEC profile is typically included in every batch-release data package because it reveals size-dependent heterogeneity in the sample. SEC profiles are considered during antibody engineering and lead candidate selection, and candidates with the cleanest profiles are chosen for subsequent drug development.

The hydrophobicity of different antibody candidates is typically compared using HIC. Increased hydrophobicity is a well-known risk factor for developability issues, which can result in off-target reactivity and poor pharmacokinetics (PK). Hydrophobicity is thus taken into account during lead candidate selection as a strategy to reduce the risk of failure due to developability issues.

Antibody-Drug conjugates (ADCs)

The drug-to-antibody ratio (DAR) and the site of drug-conjugation are important parameters for ADC characterization. HIC is able to separate molecules that differ in DAR or in the position of the drug.

Helix biosensor - SwitchSense - Dynamic biosensors for measuring binding kinetics, conformational change, bi-specific antibodies, PROTACs, molecular glue, enzyme kinetics, single cell binding kinetics - Bioanalytics with Biophysics at 2bind
Helix biosensor - SwitchSense - Dynamic biosensors for measuring binding kinetics, conformational change, bi-specific antibodies, PROTACs, molecular glue, enzyme kinetics, single cell binding kinetics - Bioanalytics with Biophysics at 2bind

Proteins and Glycoproteins

The glycan mass per glycoprotein can be determined in the triple detection system, using MALS-UV-RI. For example different glycan mass of the same glycoprotein produced in insect and mammalian cells can be detected.

In general, all parameters than can be determined for antibodies (see above) can also be measured reliably for all tpyes of proteins.

HPLC Technology and Assay Types

High Performance Liquid Chromatography (HPLC) separates molecules by using a stationary and a mobile phase. Dependent from their molecular properties as well as from the chosen stationary phase (the column material) and mobile phase (the running buffer), molecules interact with both phases and are retained within the column or move along with the mobile phase.

Eluted molecules are detected via multiple ways to allow for a most comprehensive analysis: UV-light for protein absorbance, RI (refractive index) for changes in mobile phase solution properties, and MALS (Multi-Angle Light Scattering) for precise and calibration-free molecular weight determination.

The unique characteristics of the molecules lead to differences in retention times. Molecules can be separated according to size, charge or hydrophobicity, dependent on the stationary and mobile phase, which define the assay type. 

SEC-MALS (Size-Exclusion-Chromatography)

Size exclusion chromatography (SEC) uses a gel-based column matrix. Dependent on their size and shape, molecules can enter the pores of the gel or are excluded, resulting in a prolonged retention time of smaller molecules and a short retentim time of larger molecules within the column matrix.

When SEC is coupled to a MALS detector (Multi-Angle-Light Scattering) for precise determination of molecular weight, the approach becomes SEC-MALS.

P

+ Separate the content of a sample according to molecular size.

+ Determine the molecular weight of proteins.

+ Sensitive detection of fragments and oligomers.

+ Combination with the MALS detector checks for deviations from the expected correlation of retention time and molecular weight.

O

– Contents with identical molecular weights cannot be resolved via retention time or MALS.

– Larger aggregates cannot enter the column and do not appear in the sample profile.

HIC (Hydrophobic-Interaction-Chromatography)

Reverse phase (RP) HPLC is based on a difference in the polarity of the stationary and the mobile phase and separates molecules according to their hydrophobicity. Hydrophobic molecules prefer the less polar stationary phase and are retained on the column, whereas less hydrophobic molecules elute with the more polar mobile phase. Hydrophobic interaction chromatography (HIC) is a type of RP HPLC, using a hydrophobic column matrix and separating molecules dependent on the strength of their hydrophobic interactions.

P

+ Compare the hydrophobicity of antibodies in HIC.

+ Molecular weight can be determined via MALS.

O

– Retention time is insensitive to size differences.

IEX (Ion-Exchange-Chromatography)

Ion exchange chromatography is based on electrostatic interactions between proteins and the charged column matrix, applying a gradient of increasing ionic strength in the mobile phase to elute the proteins dependent on their surface charge.

P

+ Identify charge variants in batch-to-batch comparisons.

+ Sensitive to posttranslational modifications.

O

– Requires proper assay development and selection of mobile phases.

HPLC Advantages

Helix biosensor - SwitchSense - Dynamic biosensors for measuring binding kinetics, conformational change, bi-specific antibodies, PROTACs, molecular glue, enzyme kinetics, single cell binding kinetics - Bioanalytics with Biophysics at 2bind
U

High resolution

Can resolve smaller protein fragments from monomeric proteins, as well as from higher-oligomeric structures and molecular aggregates.

High throughput

Autosampler reduces hands-on time and increases the sample throughput.

Measurement versatility

Separate or compare your samples based on size, hydrophobicity or charge.

4-parameter read-out

Besides retention time, UV absorbance, refractive index and MALS are used to describe the distribution and characteristics of molecules in your sample.

Frequently asked questions

FAQ – General

Why HPLC, not FPLC?

Whereas FPLC is the method of choice for preparative separations of proteins, HPLC provides better resolution and higher quality analytical data.

How much sample do you need for SEC-MALS and HIC?

Only 50 µg of protein are required for one HPLC run.

What is the sensitivity of SEC-MALS?

Sensitivity for impurities depends on the amount of loaded sample. Non-UV absorbing contaminations can be detected in the refractive index (RI) profile.

What is the resolution of the current SEC system?

With the current system we can separate proteins in a range of 10 to 600 kDa. Factor 2 differences in molecular weight typically result in separated peaks, smaller differences will appear as shoulder or tailing in the chromatogram.

How to interpret HIC data?

We use established positive and negative controls and use the retention time on the HIC column to rank the antibodies in relation to the controls.

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