The transition to continuous manufacturing is considered to be the next step in the biopharmaceutical industry as productivity, product quality and consistency will be significantly improved, while the environmental footprint and manufacturing costs are drastically reduced. However, a major challenge in this shift is gathering real-time information on each processing step in order to gain knowledge and control over the overall process. Thus, the development of process analytical techniques (PAT) which are able to collect critical quality attribute (CQA) information and elicit a timely response during a continuous process is then essential.<br/>Monoclonal antibodies (mAbs) may form aggregates during purification, which could lead to an increase in immune response or a decrease in efficacy. Several factors influence the formation of these species, such as pH, protein concentration or mechanical agitation, making their appearance unavoidable during continuous processing. Therefore, there is a real need to create an universal method for evaluating the formation of aggregates, by a CQA measurement for PAT use. By resorting to microfluidic biosensors placed after each purification step, these microfluidic chips will be able to work as an on-line detector, minimizing the sample volume required and providing immediate results as a PAT tool.<br/>In this work, the development of an integrated biosensing microfluidic chip for fast on-line PAT is described using hydrophobicity sensitive fluorescent dyes to examine possible size differences of mAbs species. The development and intricate design of a microfluidic structure capable to effectively mix the laminar flowing mAb sample with the fluorescent dye and subsequently collect real-time information on mAb aggregation will be presented. Firstly, the developed prototypes are validated using mAb samples with diverse levels of aggregation and two fluorescent dyes, Bis-ANS and Thioflavin T. The final prototype is then validated in a continuous chromatographic workstation on an ÄKTA™ Avant unit, operated by the control software Orbit.

Miniaturized sensor development for aggregate detection in integrated continuous processing

ACS Fall 2021

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