Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control

Figures & data

Figure 1. Experimental setup of both hardware and communication of coupling light scattering with purification system.

a). The MALS detector was connected in-line, downstream of the purification unit. HIC purified samples were also sampled downstream of the in-line MALS by on-line UHPLC. On-line UHP-SEC-µMALS served as another application to monitor the fraction and molar mass of aggregates. The samples were separated by analytical SEC and evaluated using a UHP-SEC capable multi-angle light scattering detector (µMALS) and UV signal of the on-line UHPLC as the concentration source. b) The start or inject signal and the UV signal from the purification unit were sent to the in-line MALS via an I/O Box. The UV signal of the purification unit was the concentration source used to calculate M_w of the protein eluted from the HIC column. Real time molecular weight and start/stop trigger signals were sent by the in-line MALS detector to the purification system (via a voltage signal) for fractionation when the measured M_w fell between preset M_w ranges.

Figure 2. Real time MALS data from a HIC purification.

a). The concentration and M_w signals are plotted along with the trigger signals. Throughout the purification, there is an increase in M_w (—) as aggregates saturate the HIC column and dimers coelute with the monomers. The protein is eluted from the column and passes through both the UV and in-line MALS detectors. The M_w is calculated in < 1 s using both the UV and light scattering signal and a start/stop trigger (- - -) is sent to the purification unit when the protein meets the preset M_w criteria. The concentration is calculated from the UV detector of the purification unit (---). b) No significant differences were observed between the data from real time (—) and post processed modes of analysis (---).

Figure 3. On-line UHP-SEC-µMALS data correlates well with the real time M_w data from in-line MALS.

a) Real time molecular weight (—) shown previously is rescaled to show the increase in M_w of ~ 2750 Da. The HMW content (▲) increases by 1.5% over the course of the purification as determined by on-line UHP-SEC-µMALS. b) On-line UHP-SEC chromatograms of the samples at the start and stop trigger points showed an increase in aggregate content of 1.5%. On-line UHP-SEC-µMALS data confirmed the HMW content to be dimeric (inset). The 1.5% dimeric content correlates to a M_w increase of mAb by ~ 2250 Da. The real time data showed an increase of ~ 2750 Da and is within 0.5% dimeric content of the on-line UHP-SEC-µMALS data.