CHO host cell protein assay
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Publication Number
US-11268951-B2
Publication Date
2022-03-08
Expiration Date
Abstract
The present invention provides compositions, kits, and methods for the detection of host cell proteins (HCPs) in biological samples. In some embodiments, the present invention utilizes immunization of aves hosts with proteins derived from non-aves host cells to produce aves antibodies specific for non-aves HCPs.
Core Innovation
The invention relates to detecting an amount of one or more host cell proteins (HCPs) in a sample by using HCP-specific antibodies. The sample is exposed to a first HCP-specific antibody under conditions sufficient to allow binding and to form antibody-HCP protein complexes, and the sample is then processed to remove uncomplexed HCP-specific antibodies and HCP proteins before the complexes are detected.
A key aspect is that the first HCP-specific antibody is produced in an aves host and is an IgY isotype. The antibody is capable of detecting at least 85% of the HCP proteins present in the then sample, and the disclosure emphasizes use of IgY antibodies made in avian hosts, including chickens, for HCP detection involving CHO-derived HCPs directed to non-aves host HCP proteins.
The disclosure further describes detection of antibody-HCP protein complexes in immunoassays such as ELISA and Western blot. It characterizes the IgY antibodies by comparing detection outcomes to a commercial mammalian antiserum, reporting substantially more detected HCP protein spots with the IgY antibodies and validation of an IgY ELISA with strong linearity correlation and a low quantitation limit.
Claims Coverage
The independent claim covers a two-stage detection approach based on antibody-HCP protein complex formation and detection, with an IgY isotype HCP-specific antibody produced in an aves host and required coverage of at least 85% of HCP proteins present. Three additional inventive features are recited in dependent claim coverage.
IgY HCP-specific antibody produced in an aves host for complex formation-based detection
A method of detecting an amount of one or more host cell proteins (HCPs) by exposing the sample to a first HCP-specific antibody produced in an aves host, allowing binding to form antibody-HCP protein complexes, processing the sample to remove uncomplexed antibodies and HCP proteins, and detecting an amount of the antibody-HCP protein complexes, wherein the first HCP-specific antibody is an IgY isotype and capable of detecting at least 85% of the HCP proteins present in the then sample.
Two-antibody complex detection with a labeled second HCP-specific antibody
The method further detects the amount of the antibody-HCP protein complexes by exposing the sample to a second HCP-specific antibody that binds the HCP proteins in the complexes and detecting the amount of the second antibody using a detectable label.
Detectable label options for the second antibody
The detectable label is selected from biotin, an enzyme label, or a fluorescent label.
Chicken as the aves host for IgY production
The method is carried out with a chicken as the aves host.
HCPs derived from a non-aves host
The HCPs are derived from a non-aves host.
Non-aves host restricted to selected mammals
The non-aves host is a mammal selected from hamsters, mice, rats, rabbits, or humans.
Across the independent claim and its dependent features, the coverage centers on HCP detection via antibody-HCP protein complexes using an IgY aves-produced HCP-specific antibody capable of detecting at least 85% of HCP proteins, optionally employing a second labeled HCP-specific antibody with specified label types, and narrowing the aves host and HCP source to a chicken and selected mammals.
Stated Advantages
Improved HCP detection sensitivity and specificity versus mammalian-produced antibodies.
Substantially more detected HCP protein spots compared with a commercial mammalian antiserum.
IgY ELISA validation shows strong linearity correlation and a low quantitation limit.
Documented Applications
Detecting host cell proteins (HCPs) in a sample using immunoassays such as ELISA and Western blot based on antibody-HCP protein complex detection.
Validating an IgY ELISA for CHO-derived/non-aves HCP detection using reported linearity correlation and quantitation limit.
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