The future of biopharmaceuticals looks promising with life changing treatments. The field
keeps growing and is powered by innovative, ground-breaking therapies in cancer treatment
and auto immune diseases. Advancing these novel biotherapeutics safely in the clinic requires
reliable manufacturing and quality control processes. The complex heterogenous nature of
biotherapeutics requires accurate and robust analytical testing methodologies with dependable
chromatographic separations. Identifying critical quality attributes (CQA) is the most difficult
step in implementation of quality by design (QbD) for development and production of
biopharmaceuticals. Defining each product attribute is extremely challenging and therefore,
consistency of product quality becomes even more important. We at Agilent CrossLab, designed
and manufactured our AdvanceBio columns and consumables to match our customers’ needs.
In this compendium, we have therefore selected applications to illustrate the state of-the-art
chromatographic separation for each CQA using either HPLC-UV or light scattering detectors.
We provide an overview of different chromatographic separation technologies for each CQA
using the diverse range of chemistries with in our biocolumns portfolio. There are examples
of reversed-phase, size exclusion, ion exchange, and hydrophilic interaction chromatographic
analyses of therapeutic proteins, mAbs, and antibody-drug conjugates. Let us help you improve
your productivity, method robustness, and reliability of your analytical results. We want to stand
by you on this journey in developing safer and effective biotherapeutics.
Your success is our success
Biotherapeutic proteins are highly complex molecules, which are typically produced by
fermentation using recombinant methodologies. This production process however, results in the
generation of many different variants of these proteins. Ensuring the quality of such materials
is paramount. This means confirming the product is correctly manufactured, any impurities are
identified and quantified, and the potency of the protein is determined.
As a result, it is necessary to perform tests on the intact, nondenatured molecule. Something
as large as a monoclonal antibody may contain more than 1,300 individual amino acids and
have a mass of more than 145,000 daltons. However, identifying a single minor impurity such
as deamidation of asparagine resulting in a mass difference of just one dalton, which may
occur at any of perhaps twenty or more different asparagine positions throughout the molecule,
is challenging. Only by breaking down the molecule into fragments (such as light and heavy
chains) and then into smaller polypeptide chains through enzymatic treatment is it possible to
begin to pinpoint some of these subtle differences.
Many different types of variant can be created and these are often referred to as posttranslational modifications, or PTMs. They arise after the protein has been expressed, and
can be a consequence of the manufacturing conditions, or exposure to conditions that cause
changes to occur. Fluctuations in temperature, pH, concentration, or exposure to enzymes
can all lead to variants developing. Glycosylation in particular is highly variable but is of major
importance to the efficacy of many proteins.
Understanding the different types of impurity and the risks each pose forms the basis of Critical
Quality Attribute (CQA) monitoring
The purpose of this document is to highlight some of the HPLC applications suitable for the
different aspects of CQA monitoring. As well as providing guidance for the appropriate liquid
chromatography column for the different types of detection that may be required, and to provide
a valuable reference for future consideration.
Affinity chromatography
Titer
Determination
Ideal for mAb titer determination during
process development
Glycan Analysis
Hydrophilic interaction chromatography
H2O
H2O
H2O
H2O
Fast, high-resolution, reproducible
glycan separation
Charge Variant
Analysis
Ion exchange chromatography
Enhances the accuracy and speed of
biomolecule characterization
CO2
–
CO2
–
CO2
–
Amino Acid
and Cell Culture
Analysis
Small molecule chromatography (<150 Å)
H
H H H
R O
O
N C C
Delivers robust, high-resolution separations
Intact and
Subunit Purity
Large molecule chromatography (>150 Å)
Selectivity options for every separation need
Aggregate/
Fragment Analysis
Size exclusion chromatography
Accurate, precise quantitation for a broad
range of biomolecule separations
Peptide Mapping
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