There is a growing application of integrated and continuous bioprocessing (ICB) for manufacturing recombinant protein therapeutics produced from mammalian cells. At first glance, the newly evolved ICB has created a vast diversity of platforms. A closer inspection reveals convergent evolution: nearly all of the major ICB methods have a common framework that could allow manufacturing across a global ecosystem of manufacturers using simple, yet effective, equipment designs. The framework is capable of supporting the manufacturing of most major biopharmaceutical ICB and legacy processes without major changes in the regulatory license. This article reviews the ICB that are being used, or are soon to be used, in a GMP manufacturing setting for recombinant protein production from mammalian cells. The adaptation of the various ICB modes to the common ICB framework will be discussed, along with the pros and cons of such adaptation. The equipment used in the common framework is generally described. This review is presented in sufficient detail to enable discussions of IBC implementation strategy in biopharmaceutical companies and contract manufacturers, and to provide a road map for vendors equipment design. An example plant built on the common framework will be discussed. The flexibility of the plant is demonstrated with batches as small as 0.5 kg or as large as 500 kg. The yearly output of the plant is as much as 8 tons.

An 8 ton per year manufacturing facility is described based on the framework for integrated and continuous bioprocessing (ICB) common to all known biopharmaceutical implementations. While the output of this plant rivals some of the largest fed‐batch plants in the world, the equipment inside the plant is relatively small: the plant consists of four 2000 L single‐use bioreactors and has a maximum flow rate of 13 L/min. The equipment and facility for the ICB framework is described in sufficient detail to allow biopharmaceutical companies, vendors, contract manufacturers to build or buy their own systems. The design will allow the creation of a global ICB ecosystem that will transform biopharmaceutical manufacturing. The design is fully backward compatible with legacy fed‐batch processes. A clinical production scale is described that can produce smaller batch sizes with the same equipment as that used at the commercial scale. The design described allows the production of as little as 10 g to nearly 35 kg of drug substance per day.

We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn-containing substrates. We confirmed that protein engineering did not compromise the stability of the enzyme by biophysical characterization. Comparison of wild-type ChyB and ChyB-Asn in profiling lysates of HEK293 cells demonstrated both qualitative and quantitative differences in the nature of the peptides and proteins identified by liquid chromatography and tandem mass spectrometry. ChyB-Asn enabled the identification of partially glycosylated Asn sites within a model glycoprotein and in the extracellular proteome of Jurkat T cells. ChymotrypsiN is a valuable addition to the toolkit of proteases to aid the mapping of N-linked glycosylation sites within proteins and proteomes.

We use single molecule spectroscopy to study a multicomponent, competitive protein adsorption system. Fluorescently-labeled α-lactalbumin proteins are super-resolved adsorbing to cationic anion-exchange ligands in the presence of a competitor, insulin. We find that the competitor reduces the number of binding events by blocking ligands throughout the observed measurement time while the single-site adsorption kinetics are unchanged.