Hi all,
For context, I am conducting a stability study using an ADC material formulated in different buffer systems to screen for the optimal pH/buffer to use for the final drug formulation.
The test conditions are:
- T0: freshly prepared samples
- 25C: stored for 2W and 4W
- 40C: stored for 2W
Two pH levels were tested (pH 6.3 and pH 6.8).
At the test points (T0 and 2W) the samples were collected and frozen in -80C to perform the analysis altogether for all samples at 4W. 4W samples were also frozen for 2 hours prior to testing in order to give a uniform treatment for all the samples.
At 2W, all samples (25C and 40C) were similarly stable and comparable.
At 4W (25C), all samples except for one were comparatively stable and show similar patterns despite using different buffer systems or pH levels.
Usually, at 25C all samples remain stable even at Week 4. Severe changes are usually observed at the higher temperature and longer time point (ie. 40C, Week 4).
The analyses performed were:
> SEC-HPLC for monomer purity
> HIC-HPLC for DAR value
> CE-SDS (NR and R)
The observations with the irregular sample were:
Appearance:
> The solution is clear and colorless, but there is a visible white agglomerate that seems to float around.
SEC-HPLC:
> The monomer peak area and height is visibly decreased, and several LMW peaks were observed (not present in the other samples). [attached a figure file]
HIC-HPLC:
> DAR4 peak is highly decreased (even lesser than the W2-40C sample), DAR2 peak appears very broad, plus the appearance of several low hydrophobicity peaks and a few peaks after the DAR4 peak which were not present in all of the other samples. [attached a figure file]
CE-SDS NR:
> 2H2L peak is decreased around 2-fold and 2H1L peak is increased 5-fold.
Questions:
1) Based on the observation, is this phenomenon protein degradation or contamination?
2) How to prove either of these assumptions?
> I personally lean towards protein degradation, but some think it might be contamination.
3) Can anyone share any similar experiences and how you interpreted it?
I look forward to discussions and thanks in advance!
1) Based on the observation, it seems more likely that the phenomenon is protein degradation rather than contamination. Protein degradation can result in the formation of aggregates, low molecular weight fragments, changes in charge variants, and alterations in the drug-to-antibody ratio (DAR) ¹. Contamination, on the other hand, would introduce foreign substances or microorganisms that might affect the purity, potency, or safety of the protein ². The appearance of a white agglomerate, the decrease in monomer peak and DAR4 peak, the increase in LMW peaks and 2H1L peak, and the broadening of DAR2 peak are all indicative of protein degradation ¹³⁴.
2) To prove either of these assumptions, you can perform additional tests to confirm the presence or absence of degradation products or contaminants. For example, you can use size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to measure the molecular weight and aggregation of the protein . You can also use mass spectrometry (MS) to identify the degradation products and their modifications . For contamination, you can use microbiological assays to detect any microbial growth or endotoxins . You can also use polymerase chain reaction (PCR) to detect any DNA contamination .
3) I have not personally experienced a similar situation, but I found some case studies that might be relevant to your problem. One case study reported the degradation of an ADC due to hydrolysis of the linker between the antibody and the drug . Another case study reported the degradation of an ADC due to oxidation of the antibody . Both cases showed similar changes in the SEC-HPLC, HIC-HPLC, and CE-SDS profiles as your sample. You can read more about these case studies in the links provided.
(1) Mechanisms of Protein Degradation - Thermo Fisher Scientific. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/protein-degradation-resource-center/mechanisms-protein-degradation.html.
(2) Mechanisms of Protein Degradation - Taylor & Francis Online. https://www.tandfonline.com/doi/pdf/10.4161/cc.4.11.2115.
(3) How is the balance between protein synthesis and degradation achieved .... https://tbiomed.biomedcentral.com/articles/10.1186/1742-4682-7-25.
(4) undefined. http://www.landesbioscience.com/journals/cc/abstract.php?id=2115.
Raghad Mouhamad thanks for your detailed response and for the links you shared.
It seems evident that this is a case of protein degradation based on the LC and CE profiles, but do you agree that this phenomenon could also be caused or triggered by a "contaminant" (eg. fungal or bacterial particle or other particulate matters)?
I am still a newbie in the biologics formulation field so I have actually a lot of questions regarding this.
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