CYGNUS Technologies anticipate our current supply of capture and detection antibodies used in Catalog # F650S HEK 293 HCP ELISA Kit 3G will be depleted by the end of calendar year 2021. This is due to an unforeseen demand for F650S kits to support major global COVID-19 adenovirus-based vaccine programs as well as continuing strong growth in the number of cell and gene therapy development projects conducted by our customers. In response to this demand, Cygnus Technologies produced and characterized new antibodies for an ELISA format to match F650S kit performance as close as possible. The new antibodies have similar coverage to HEK 293 HCPs in the control HEK 293 antigen (Lot # 170320) as determined by Antibody Affinity Extraction with 2D PAGE/Silver Stain and LC-MS.
|Method||F650S Coverage %||Resupply 1 F650S Coverage %|
|AAE™ with 2D-PAGE||89%||90%|
Furthermore, comparative analysis of both antibodies by AAE-MS demonstrated that 92% of immunoreactive HCPs in the control HEK 293 antigen (Lot # 170320) are similar between F650S and Resupply 1 F650S antibodies.
Kits containing the new antibodies will maintain the same catalog number, F650S, and will be identified by the lot numbers starting with characters “R1”. This notification is provided to your company in advance to allow time to plan and perform any bridging, re-qualification, or re-validation studies that you may deem necessary for transition to the new HEK 293 HCP antibody and corresponding F650S HEK 293 HCP ELISA Kit 3G Resupply 1.
The following table compares F650S and Resupply 1 F650S assay design and specifications and highlights changes:
|F650S||Resupply 1 F650S|
|Capture Antibody||Goat + Rabbit||Goat + Rabbit|
|Antigen||A mild lysate of HEK 293 cells extracted by a procedure similar to those used in propagating and harvesting cells producing viral vectors, followed by antigen processing using Cygnus proprietary method aimed to generate a broad immune response||A mild lysate of HEK 293 cells extracted by a procedure similar to those used in propagating and harvesting cells producing viral vectors, followed by antigen processing using Cygnus proprietary method aimed to generate a broad immune response|
|Affinity Purified||Yes||Yes, identical procedure|
|Antibody coverage analysis as determined by AAE with 2D-PAGE||74% (Lot # 60619)||90% (Lot # 170320)|
|Antibody coverage analysis as determined by AAE-MS||n/a||91% (Lot # 170320)|
|LOD||0.2 ng/ml||0.35 ng/mL|
|LOQ||1 ng/mL||1 ng/mL|
|Precision||Intra-assay: 4.2 – 7.1 %CV
Inter-assay: 2.6 – 5.2 %CV
|Intra-assay: 4.2 – 5.0%
Inter-assay: 4.3 – 6.6%
|Accuracy||90 – 99%||89 - 113%|
|Detection Ab and Sample/ Standard/Control incubation time||1.5h||1.5h|
|Shelf Life||9 months||9 months|
|Example Kit Lot Number||260521-4||R1260521-4|
Cygnus Technologies has evaluated these new antibodies in the ELISA using a panel of 13 process samples obtained from several customers. Most of the samples in our test panel gave less than a 2-fold difference between antibodies, some samples showed differences of up to 7-fold between the two assays. There was no trend for consistently higher or lower values in one assay or the other.
We suggest at least the following studies be performed to qualify the Resupply 1 F650S kit and corresponding capture antibody:
- Establish the mean and acceptable range for your controls with the Resupply 1 F650S kit. These values may be different (higher or lower) relative to the current antibody. To avoid failing runs due to controls ‘out of specification’ it may be necessary to set a new range. If you use other curve parameters such as ODs as an indirect specification, these too may require a new range to be established.
- Test in-process and drug substance samples using the F650S and Resupply 1 F650S kits in parallel to determine if there is a consistent and significant difference and bias (higher or lower values) from the F650S kit.
- Perform dilution linearity and spike recovery on your samples with the new antibodies to assure accuracy and specificity.
- Orthogonal determination of coverage is best determined using our AAE method. We recommend performing AAE on at least two samples: 1) an upstream harvest sample to determine coverage to the majority of the proteome and 2) a down-stream sample to determine coverage those HCPs that persist through the purification process. Cygnus can perform the AAE analysis for you. Please note: Cygnus is offering a 20% discount on all AAE analysis requested in support of this change.
- If you are using the F650S kit for lot release testing, determine what if any effect differences in control and sample values will have on your release criteria and document those changes.
Kits using the new antibody are now available, so that you can begin comparison studies. New kits are distinguished from old kits by the Lot Number starting with “R1”. The current F650S kits will be shipped until our supplies are exhausted.
To order new kits, please specify catalog # F650S Resupply 1 on the purchase order and send an email to firstname.lastname@example.org.