Comprehensive Analytics Solutions
|Protein Analytics||Quality Attribute||Platform|
|Intact Mass (non-reduced, reduced, +/-PNGaseF)||Primary Structure/PTMs||LC-MS/QTOF|
|N – Glycan profiling||Primary Structure/PTMs||LIF/CE-SDS/cIEF/CZE|
|Sialic Acid Content||PTMs||FLD/UHPLC|
|N -Glycan profiling (HILIC)|
|Tm, Tagg, Size and PDI||Thermal Stability/Higher Order Structures||SLS/DLS|
|ELISA||Screening/Potency/Activity||Multimode plate reader|
|Binding kinetics (kon, koff, KD)||Potency/Screening/Selectivity||BLI or SPR|
|FCR and C1Q screening|
|Yes /No binding|
|IEX||Purity/Higher Order Structures||HPLC|
|MW by MALS|
|Host Cell Proteins (Cygnus)||Safety||ELISA|
|Residual Protein A/G/L|
Developability is a term often used to describe the evaluation process applied to lead molecules for their potential to be developed into drug products. Developability studies encompass a rigorous set of biochemical and biophysical tests to determine the candidate’s critical quality attributes (CQA) that are necessary for functionality and stability. This process allows the investigator to identify candidates to move forward and exclude/redesign those with unfavorable profiles, thus avoiding potential pitfalls during the lifecycle of a biotherapeutic. At Aragen Bioscience, we provide a suite of services available for the customer to identify their CQA and test how those attributes are susceptible to a particular stress, see below.
The stability of any lead candidate(s) is a requisite for its potential biotherapeutic application. An approach to assess the integrity of the molecule is to perform a heat-stress study that can accelerate and highlight any instability issues that may be present. For the example below, two molecules were lead candidates in a study that underwent a number of stressors prior to proceeding in the developability pipeline. The heat-stress data displayed stark differences in their stability profiles.
Lead candidates were subjected to a 28-day heat stress at 40°C followed by charge variant (cIEF) and size variants (CE-SDS) analytics. The heat stress study demonstrated that both molecules undergo changes in their charge variants (Figure 1). Comparing Day 0 (black traces) to Day 28 (red traces) the main peak species for both molecules decreased and shifted to more acidic species (increase in the distribution of acidic peaks), suggesting that oxidation and deamidation are likely occurring. This is observed in both molecules, but to a greater extent on lead candidate #1 suggesting that this molecule is more susceptible to heat stress than candidate #2.
Using size variant (CE-SDS) analysis in both reducing (Figure 2) and non-reducing (Figure 3) formats, we observed that the main peaks for candidate #1 diminish more extensively than candidate #2. Specifically, candidate #1 main peak in the reduced format shows that there are additional species or fragments present on Day 28 that are not present on Day 0 (Figure 2, left). This is further supported by non-reducing CE-SDS (Figure 3, left panel) where the intact main peak at Day 0 has deteriorated by Day 28 at 40°C suggesting fragmentation of both light and heavy chains. Contrastingly, lead candidate #2 demonstrated minimal fragmentation retaining the overall size variant profile at Day 28 as was present at Day 0 (Figures 2 and 3, right panels). In conclusion, the advancement of lead candidate #2 suggests having a higher likelihood of success through the development process, where oxidation or deamidation may be mitigated through the addition of excipients.
Figure 1. Charge variant (cIEF) analysis of two lead molecules demonstrates the effects of a heat stress study.
The cIEF electropherograms for lead candidate #1 (left) and #2 (right) at Day 0 (black) and after 28 days at 40°C (red). On Day 0, the lead candidate #1 abundance of the main peak (~25.3-25.7 min) with a pI of 8.7 is ~75% then drops to ~30% by Day 28. For lead candidate #2, the abundance of the main peak (~21.4-21.6 min) with a pI of 9.5 is ~79% then drops to ~63% by Day 28. Results were collected on a Sciex PA800 Plus system, analyzed using the 32Karat software, and figures generated using MATLAB (R2019b).
Figure 2. Reducing size variant (CE-SDS) analysis of two lead molecules demonstrates the effects of a heat stress study.
The reducing CE-SDS electropherograms for lead candidate #1 (left) and #2 (right) at Day 0 (black) and after 28 days at 40°C (red). On Day 0, the lead candidate #1 abundance of the main peaks comprising of light (~19 min) and heavy (~24 min) chains was ~97%, but drops to ~53% by Day 28. Notably, there are fragments in between the light and heavy, indicating fragmentation of the heavy chain. For lead candidate #2, the abundance of the light (~19 min) and heavy (~24 min) chains remain high starting ~99% then drops to ~97% by Day 28 suggesting the molecules remain intact throughout the stress study. Results were collected, analyzed, and figures generated as noted in the legend of Figure 1.
Figure 3. Non-reducing size variant (CE-SDS) analysis of two lead molecules demonstrates the effects of a heat stress study.
The non-reducing CE-SDS electropherograms for lead candidate #1 (left) and #2 (right) at Day 0 (black) and after 28 days at 40°C (red). On Day 0, the lead candidate #1 abundance of the main peaks comprising of the intact molecule (~34 min) starts at ~96% but drops to ~7% by Day 28. Unfortunately for the lead candidate #1, the fragmentation (~16-33 min) of the main species is observed by Day 28. For lead candidate #2, the abundance of the intact molecule (~34 min) remains high starting ~92% then drops to ~88% by Day 28 suggesting the molecules remain intact throughout the stress study. Results were collected, analyzed, and figures generated as noted in the legend of Figure 1.