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For personalized medicines, national emergencies, military operations, and related applications there is a growing need to produce single-dose levels of therapeutic proteins at the point of care. To satisfy this need, the ?biologically-derived medicines on demand? (Bio-MOD) device has been developed, which is an integrated and portable continuous bioprocessing system. For the upstream processing of this device, a cell-free expression system is utilized which provides less than 0.5% purity in the harvest. For the downstream processing component of this device, an intensified method is required not only to provide more than a 10,000-fold host cell protein (HCP) reduction factor, but also to offer a fast, simple, and cost-effective separation. The focus of this thesis is the development of an intensified downstream process for the Bio-MoD that is based on either a mathematical or a chemical approach. For the mathematical approach, a 3-D fine-grained finite-element mechanistic model is developed using COMSOL Multiphysics for the chromatography processes of the Bio-MoD device. The model is validated using both analytical and experimental methods and can efficiently explore the parameter space affecting the performance of the chromatography processes. For the chemical approach, an affinity capture method implemented with a simple design of experiment procedure is used as a generic element of the purification method for the Bio-MOD device. In order to produce a biosimilar without any additional tag on the product, the intensified separation technique of chromatofocusing optimized using computer simulations is employed as a non-affinity capture step. The results obtained show that, through the use of computer simulations, a non-affinity capture step can be made sufficiently ?generic? to be a useful alternative for the Bio-MOD device. Finally, the concept of ion-exchange (IEX) chromatofocusing is applied on an immobilized metal affinity chromatography (IMAC) column in order to improve the purity by performing polishing at the same time as the capture step.

Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the HO cell-free system. However, many complex proteins are still difficult to express using this method. To investigate the current bottlenecks in cell-free glycoprotein production, we chose erythropoietin (40% glycosylated), an essential endogenous hormone which stimulates the development of red blood cells. Here, we report the production of recombinant erythropoietin (EPO) using CHO cell-free system. Using this method, EPO was expressed and purified with a twofold increase in yield when the cell-free reaction was supplemented with CHO microsomes. The protein was purified to near homogeneity using an ion-metal affinity column. We were able to analyze the expressed and purified products (glycosylated cell-free EPO runs at 25–28 kDa, and unglycosylated protein runs at 20 kDa on an SDS–PAGE), identifying the presence of glycan moieties by PNGase shift assay. The purified protein was predicted to have ∼2,300 IU in vitro activity. Additionally, we tested the presence and absence of sugars on the cell-free EPO using a lectin-based assay system. The results obtained in this study indicate that microsomes augmented in vitro production of the glycoprotein is useful for the rapid production of single doses of a therapeutic glycoprotein drug and to rapidly screen glycoprotein constructs in the development of these types of drugs. CFPS is useful for implementing a lectin-based method for rapid screening and detection of glycan moieties, which is a critical quality attribute in the industrial production of therapeutic glycoproteins., http://onlinelibrary.wiley.com/doi/10.1002/bit.26554/full?wol1URL=/doi/10.1002/bit.26554/full&regionCode=US-MD&identityKey=cdcb031c-9694-4db7-9d62-8b1333523f4c