ATUM has extensive in-house assay capabilities,
including mass spectrometry, HPLC, fluorescent reader
and bio-layer interferometry (ForteBio Octet Red384
and HTX Systems).
We can develop a new assay to support your project, or transfer
your assay to us. If you have a specialized assay that needs to
be performed by you or by a 3rd party, we will work with you
to develop appropriate procedures for material and data transfer.
our assay development offerings
Below are select examples of assays that we have
developed and performed for clients. We can help
develop a new assay to support your project, or
transfer your assays to us.
Functionality
Antibody Affinity
Enzyme Specificity
Enzyme Activity
Developability
Expression
Solubility
Aggregation
Stability
Post-translational modifications
Antibody Affinity
Antigen-antibody interactions are non-covalent and reversible, formed by a combination of
hydrogen bonds, hydrophobic interactions, electrostatic and van der Waals forces. We can
measure antibody affinity, i.e. the strength of the interaction between an epitope and an
antibody’s antigen binding site by ELISA or by using our Octet Biosensors system. Our Octet
systems also provide the ability for kinetic characterization. Antibody affinity for
an IgG measured with our Octet system is shown.
The figure below shows an affinity screen with full kinetic characterization of Fab-ligand interaction.
Antigen was covalently cross-linked to the Octet sensor via an amine linkage. Antigen coated sensors were placed into
a Fab solution at a defined concentration for 300 seconds. Binding of the Fab to the antigen on the tip resulted in an
increase in the Octet signal, which was proportional to the amount of Fab bound. The sensors were then moved to buffer
for 300 seconds and the dissociation of the Fab from the antigen was measured by a decrease in Octet signal as the
Fab fell off the tip. The ratio of dissociation rate (koff) to association rate (kon) gives an estimate
of the dissociation constant (Kd) as a measure of antigen-antibody affinity.
Enzyme Specificity
One of the properties of enzymes that makes them so important is the specificity they
exhibit relative to the reactions they catalyze. Traditionally, we support enzymatic
specificity studies with different substrates performing individual kinetics measurements
using fluorescence spectroscopy. We can also multiplex this approach using electrospray ionization mass
spectrometry to quantify multiple products simultaneously. If the specificity of an enzyme
does not meet the requirements needed, we can improve this attribute using
our protein GPS technology.
Homolog
Enzyme specificity against 4 substrates was measured as a continuous kinetic colorimetric
readout using absorption spectroscopy. A client asked ATUM to engineer an enzyme for activity
against 4 substrates but the proposed starting point was active against only one substrate. We were
able to discover several homologs with significantly increased activity against all 4 substrates.
Enzyme Activity
Enzyme activity is a measure of catalytic ability which can be measured by the decrease in a
substrate concentration during a set period or to measure the increase in the concentration
of a product after some time. We can measure the course of an enzymatic reaction by absorption
and fluorescence spectroscopy.
Homolog
Enzyme activity against 4 substrates was measured as a continuous kinetic
colorimetric readout using absorption spectroscopy.
Expression
ATUM has extensive experience in optimizing the soluble expression of proteins in mammalian,
bacterial and yeast host. We have an extensive catalog of vectors and vector elements that can
be used to express a protein at the desired levels and in specific cell types. Working with
you, we can develop or provide expression vectors particular to a defined need. In
conjunction with our codon optimization tools, we offer unparalleled opportunities
to express your proteins at desired levels in your chosen host or cell tissue.
Different promoters work better for different proteins. Expression of three proteins,
phi29 (~61 kD), cutinase (~22 kD) and DasherGFP (~26 kD), under control of different
promoters, T5 and T7 (IPTG-inducible), rham (rhamnose-inducible) and phoA
(inducible by phosphate starvation) is shown on an SDS-PAGE gel.
Solubility
The solubility of proteins is determined by various interactions including protein-protein,
protein-water, protein-ion, and ion-water. The extremes of protein solubility can range from
almost complete insolubility to several hundred of milligrams per milliliter. We can
test the solubility of a protein by measuring the protein concentration in solution after
centrifugation. Further testing also includes HPLC-SEC to determine whether the protein has
started to aggregate when testing whether high concentrations of the protein are soluble and
SDS-PAGE analysis to determine whether the protein has degraded. Strategies for increasing in
vitro protein solubilities include studying which buffers or additives can stabilize a protein,
which we study using a thermal shift assay. Alternatively, it is possible to increase
protein solubility using our protein GPS approach replacing surface hydrophobic residues
with hydrophilic residues or fusing highly soluble peptides or proteins to the target protein.
Soluble protein expression using different solubility tags is protein dependent. Three proteins
encoded by genes A (~48 kD), B (~49 kD) and C (~59 kD) with solubility problems were expressed in
E.coli expression vectors (pD441-XXX) with various N-terminal solubility tags MBP (~42 kD),
GST (~26 kD), PpiBT (~19 kD), Fh8 (~8.8 kD) and 6XHis as a negative control (~1 kD) under control of
the IPTG-inducible T5 promoter, at 37°C and room temperature (RT). Higher soluble protein expression
was observed at RT except for gene A with MBP tag. BL21 cells were transformed and grown at 37°C in
2 ml LB + 30 µg/ml kanamycin to an OD600 of 0.8. Cultures were induced by addition of 1mM IPTG and
incubated for 3 hrs at 37°C or overnight at room temperature (RT); uninduced cultures were run as
negative controls. Samples were pelleted, lysed and total and soluble preps prepared. Total protein
(T) samples and soluble fractions (S) were denatured and reduced in sample buffer with reducing agent
at 95°C for 10min. 5µl (approx. 5 O.Ds) of denatured sample was loaded per lane of a 4-12%
NuPAGE Bis-Tris MES gel and Coomasie stained. Band volumes (shown in red boxes) were measured and
quantitated using BSA as a reference.
Aggregation
Aggregates are typically large, tangled clusters of denatured protein molecules that are
irreversibly formed either during product expression in cell culture, product purification
in downstream processing, or storage as drug substance or drug product. Our HPLC-SEC assay
allows for aggregation formation to be monitored throughout our process which can be addressed
by removal during purification. If a protein aggregates excessively we can use our ProteinGPS
engineering technology to optimize the ability of the protein to stay in a monomeric state while
maintaining function.
A Cytokine was purified using a single step purification method, and desalted into an appropriate
formulation buffer prior to being run on a HPLC-SEC column. The UV trace from the Cytokine product
shows multiple peaks corresponding to the product, impurities and possibly higher aggregates.
Cytokine / HPLC / SEC Agilent
Peak #
Peak Retention Time (min)
Area %
SEC Calculated MW (kDa)
Peak Assignment
1
5.60
2.40
144.9
Higher aggregates
2
7.921
93.87
12.2
Product
3
9.930
2.59
1.4
Impurity
4
10.984
1.14
0.5
Impurity
Stability
Protein Stability is the balance of forces that determine whether a protein is in its native
folded conformation or a denatured (unfolded or extended) state. A common goal is the creation
of an environment in which protein samples can stably retain native conformations.
Tm is the proteins melting temperature and is a reporter of the protein's thermal stability. We use the
thermal shift assay that uses a fluorescent dye to monitor the temperature dependent unfolding of a
protein or the denaturation temperature performed on a quantitative PCR system as a high throughput
screen.This can be used to screen conditions that affect protein thermal stability, such as protein
mutations, ligand binding, and buffer formulations (for example pH, salts, detergents and other
additives). The example below shows Tm measurements for a subset of 10 variants from a 96 protein
variants screen.
Post-translational modifications
Many biological therapeutics being developed today, have or are at risk from potential
Post-Translational Modifications (PTMs). These structural protein modifications are considered relevant
to determining a molecule’s overall long-term stability, safety, and efficacy. Our HPLC and
Mass Spectrometry and micro CE systems provide the ability to study charge, hydrophobic and glycan variants.
Furthermore, we can determine the mass of the molecule and by peptide mapping determine the
location of each modification.
Glycosylation profile of a therapeutic Mab was measured using micro CE. The microchip based CE analysis gives resolution
sufficient to determine the relative abundance of the major N-glycan types found on antibodies,
offering a high throughput option.
µCE / Glycan analysis Herceptin
N-glycosyation of therapeutic proteins can be monitored by the enzymatic N-glycan release followed by
labeling with a fluorescent tag to run HILIC-FLD analysis. The Zoomed in FLD chromatogram is of a
N-Glycan sample from mAb Herceptin showing detection of major and minor glycans. The relative
abundance of each glycan is shown in the table below.
HPLC Glycan analysis Herceptin
µCE / Charge Variant Herceptin
Microchip based CE was used to determine the charge profile of mAb Herceptin. The profile of this particular mAb includes two basic variants, one main variant and three acidic
variants. The resolution of the assay is sufficient to determine the relative abundance of each
variant.
Have a question? Let's talk.
ATUM customer support scientists are available to discuss cloning strategies,
gene design constraints, bioinformatics analyses, and other molecular
biology/biotechnology concerns.
