HPLC Analysis Related Questions
CellMosaic offers a variety of HPLC analysis and method development for our customers. For general information regarding HPLC analysis, please click here. The following lists some Frequently Asked Questions regarding HPLC analysis.
How many samples do I need to supply for ADC HPLC analysis per conjugation?
We recommend providing two (2) samples: the ADC and unlabeled antibody for comparing the profile (e.g. how much unlabeled antibody remains in the ADC, and how much aggregation ADC may add).
I have only a small amount of conjugate to spare. What is the minimum amount of sample that I need to provide for SEC and HIC HPLC analysis?
For SEC HPLC analysis of an ADC, if you only need to check the aggregation profile and concentration, we can use as little as 2 µg per injection. For HIC, we need a minimum of 10 μg per injection. So, if you can send us 20 µL of the ADC solution (1 mg/mL), we can do the analysis.
If you have any questions about these amounts, please contact us and we can give you an expert recommendation on how much you need to send us and what analysis we believe to be necessary.
Is there a setup fee for HPLC analysis?
Depending on the effort of setting up, each type of HPLC analysis may require a setup fee. However, the setup fee will be waived if the conjugate is made using CellMosaic’s kit or if the sample can be analyzed within a longer timeframe of up to 7 business days. The standard service is 1-3 business days. If you need the sample analyzed right away, a setup fee will be added.
What would the price be for a bulk order HPLC analysis?
If the total number of samples is over 20, you will get a 5% discount for each sample. If it is over 31, you will get a 10% discount per sample. If you need the sample analyzed right away, a setup fee per batch analysis will be added.
How soon after I send in my samples for analysis should I see results?
1-3 days on average unless you are told otherwise.
Why does the baseline for my HIC data seem to fluctuate a little more than usual?
We often use 220 nm for antibody and protein detection due to the limited amount of sample we use for analysis. However, the HIC salt/buffer we use has absorbance at 220 nm. With the salt gradient we are running, the blank absorbance at 220 nm can fluctuate a bit depending on the equipment/environment. If the absorbance of your biomolecule at 220 nm is low, or the actual concentration is lower than the given concentration, you are more likely to obtain results with more apparent fluctuations in the baseline.
I noticed that your company offers DAR analysis using HIC. Could you please let me know the price per sample? In addition, do you also measure free MMAE in the ADC?
$100 per sample and $250 setup fee. The setup fee is waived if the conjugate is made using CellMosaic’s kit (as of June 2024). You will need to provide two (2) samples (unlabeled and labeled ADC) per labeling. The HIC data will tell if there is any free MMAE left. In general, if you use our kit, there will be no free MMAE. Please let us know how many unlabeled antibodies and labeled antibodies you need to analyze when inquiring further.
One of my antibodies is IgM. Is it okay to confirm DAR using HIC?
We have done some IgM conjugation and HIC analysis. Due to the huge MW of the IgM, the HIC may not have the separation needed to calculate the DAR.
Can we use a different column from Waters for HIC HPLC?
Yes, the HIC HPLC separation principle is the same. The difference is what particle they use, the efficiency/density of the capping, particle size, and column width and length. You will have to find out if they are suitable or not.
Can we use Agilent HPLC 1200 for HIC analysis?
Yes, you can use HPLC 1200 equipment. Just make sure to wash your HPLC equipment thoroughly after finishing each analysis (step 9 on the product sheet). Perform the routine HPLC maintenance more often if you are using these high salt buffers.
Where can I find the conditions and buffers for HIC analysis?
CellMosaic sells a high quality HIC buffer set (Cat#: CM02025) for convenient analysis of bioconjugates. You can find the buffer compositions and HIC HPLC run conditions from the product sheet on our website.