End-to-end collaboration to transform biopharmaceutical development and manufacturing.

An ambitious 10-year collaborative program is described to invent, design, demonstrate, and support commercialization of integrated biopharmaceutical manufacturing technology intended to transform the industry. Our goal is to enable improved control, robustness, and security of supply, dramatically reduced capital and operating cost, flexibility to supply an extremely diverse and changing portfolio of products in the face of uncertainty and changing demand, and faster product development and supply chain velocity, with sustainable raw materials, components, and energy use. The program is organized into workstreams focused on end-to-end control strategy, equipment flexibility, next generation technology, sustainability, and a physical test bed to evaluate and demonstrate the technologies that are developed. The elements of the program are synergistic. For example, process intensification results in cost reduction as well as increased sustainability. Improved robustness leads to less inventory, which improves costs and supply chain velocity. Flexibility allows more products to be consolidated into fewer factories, reduces the need for new facilities, simplifies the acquisition of additional capacity if needed, and reduces changeover time, which improves cost and velocity. The program incorporates both drug substance and drug product manufacturing, but this paper will focus on the drug substance elements of the program.

Pilot-scale demonstration of an end-to-end integrated and continuous biomanufacturing process.

There has been increasing momentum recently in the biopharmaceutical industry to transition from traditional batch processes to next-generation integrated and continuous biomanufacturing. This transition from batch to continuous is expected to offer several advantages which, taken together, could significantly improve access to biologics drugs for patients. Despite this recent momentum, there has not been a commercial implementation of a continuous bioprocess reported in the literature. In this study, we describe a successful pilot-scale proof-of-concept demonstration of an end-to-end integrated and continuous bioprocess for the production of a monoclonal antibody (mAb). This process incorporated all of the key unit operations found in a typical mAb production process, including the final steps of virus removal filtration, ultrafiltration, diafiltration, and formulation. The end-to-end integrated process was operated for a total of 25 days and produced a total of 4.9 kg (200 g/day or 2 g/L BRX/day) of the drug substance from a 100-L perfusion bioreactor (BRX) with acceptable product quality and minimal operator intervention. This successful proof-of-concept demonstrates that end-to-end integrated continuous bioprocessing is achievable with current technologies and represents an important step toward the realization of a commercial integrated and continuous bioprocessing process.

Dynamics differentiate between active and inactive inteins.

The balance between stability and dynamics for active enzymes can be somewhat quantified by studies of intein splicing and cleaving reactions. Inteins catalyze the ligation of flanking host exteins while excising themselves. The potential for applications led to engineering of a mini-intein splicing domain, where the homing endonuclease domain of the Mycobacterium tuberculosis RecA (Mtu recA) intein was removed. The remaining domains were linked by several short peptides, but splicing activity in all was substantially lower than the full-length intein. Native splicing activity was restored in some cases by a V67L mutation. Using computations and experiments, we examine the impact of this mutation on the stability and conformational dynamics of the mini-intein splicing domain. Molecular dynamics simulations were used to delineate the factors that determine the active state, including the V67L mini-intein mutant, and peptide linker. We found that (1) the V67L mutation lowers the global fluctuations in all modeled mini-inteins, stabilizing the mini-intein constructs; (2) the connecting linker length affects intein dynamics; and (3) the flexibilities of the linker and intein core are higher in the active structure. We have observed that the interaction of the linker region and a turn region around residues 35-41 provides the pathway for the allostery interaction. Our experiments reveal that intein catalysis is characterized by non-linear Arrhenius plot, confirming the significant contribution of protein conformational dynamics to intein function. We conclude that while the V67L mutation stabilizes the global structure, cooperative dynamics of all intein regions appear more important for intein function than high stability. Our studies suggest that effectively quenching the conformational dynamics of an intein through engineered allosteric interactions could deactivate intein splicing or cleaving.

Purification of E. coli proteins using a self-cleaving chitin-binding affinity tag.

The use of affinity tags to purify recombinant proteins is ubiquitous in molecular biology. However, tag removal after purification still remains a challenge. The most commonly used method, proteolytic digestion, has several drawbacks that make the process complex and costly. One alternative to the use of proteolytic digestion is the use of self-cleaving purification tags. Here, we describe a system that combines a chitin-binding domain (CBD) tag with the ∆I-CM intein to yield a self-cleaving purification tag. A protein gene of interest is genetically fused downstream of the tag, generating a fusion protein that can be rapidly and easily purified using a chitin resin. Intein self-cleavage is then induced by a simple pH and temperature shift, liberating the free target protein. This system can be used to readily purify any recombinant protein that can be expressed in E. coli, and has the potential to be applied to a wide variety of additional tags and expression hosts.

Ligation-independent cloning and self-cleaving intein as a tool for high-throughput protein purification.

The rapid production of purified recombinant proteins has become increasingly important for countless applications. Many purification methods involve expression of target proteins in fusion to purification tags, which often must be removed from the target proteins after purification. Recently, engineered inteins have been used to create convenient self-cleaving tags for tag removal. Although intein methods can greatly simplify protein purification, commercially available expression vectors still rely on conventional restriction/ligation cloning methods for target gene insertion. We have streamlined this process by introducing Ligation-Independent Cloning (LIC) capability to our intein expression plasmids, which provides a simple method for constructing self-cleaving tag-target gene fusions. In this work, we demonstrate efficient gene insertion via this system, as well as target protein expression and purification consistent with previously reported results. Through this newly developed system, arbitrary protein genes can be rapidly incorporated into self-cleaving tag expression vectors, and their products purified using convenient platform methods.

Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.

In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain-intein tag for purification via a chitin-agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and ß-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the ΔI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.