November 15, 2016 (Vol. 36, No. 20)
Novel Solutions for Recombinant Protein Production
While much has been said about the rise of biologic-based therapeutics, less has been said about the challenges that stand in the way of producing them reliably and economically. In the initial phase of the process, the challenges lie mainly in integrating expression constructs in mammalian cellular systems stably and at high expression levels, while achieving the correct processing of desired protein products.
One such tool that has been shown to be faster, more reliable, and more productive than conventional methods (i.e., random integration and selection of expression constructs) is the piggyBac™ transposon system (PB). This nonviral technology addresses the aforementioned challenge of generating stably transfected mammalian cell cultures in protein production applications.
The PB system functions via a “cut and paste” mechanism (Figure 1), which inserts the transposon carrying a transgene, at semi-random TTAA sites (skewed toward more accessible, transcriptionally active sites) throughout the genome.
In this tutorial, we’ve gathered key findings from recent research to highlight the advantages of the piggyBac system to generate therapeutic proteins. These studies further affirm the ability of the system to boost productivity and workflow efficiency in the crucial early phases of biotherapeutic development.
Figure 1. The piggyBac transposon system. The desired cargo (i.e. a recombinant protein) is cloned into the piggyBac transposon vector, between the inverted terminal repeats (ITRs). The transposon vector and transposase are both transfected into the cell line. Expression of the transposase leads to recognition of the transposon within the vector and eventually integration.
Shorter Timelines with piggyBac
For decades, the rule for new therapeutic development has been to fail fast. Since late-stage failures are extraordinarily costly, it is more cost-effective to eliminate candidates as early as possible. To deliver quickly on gram quantities of protein for testing in an appropriate model system, researchers cannot always rely on traditional, time-consuming single cell cloning to identify high expressing clones, therefore alternate strategies must be employed. Initial preclinical manufacturing runs of recombinant proteins often include numerous variations to identify the best candidate to move forward.
These initial batches can be done as transiently transfected pools of cells to circumvent the time-consuming clonal isolation of cells. Only once the final candidate molecule has moved to the clinical stage is the more laborious single cell clone required.
Strategies to utilize pools of cells, either through transient expression or stable, random plasmid integration, have been used. While this can shorten timelines, significant pitfalls remain due to lack of stability and reproducibility. Using the PB system, from transfection to g/L productivity in a one-liter scale is achievable in as little as four weeks. Compared to older, nontransposon based systems, this advancement allows for ease of scale-up with frozen banks for repeated production runs, requires much less DNA and shows stable expression for at least three months without continued selection.
Furthermore, if a single cell clone is required, the likelihood of finding a sufficiently high-expressing clone is significantly higher (see below), requiring less hands on time dedicated to screening clones and further reducing costs.
Increased Recombinant Protein Titer
Given the amount of time and cost invested in the development of new biopharmaceuticals, the need to express large quantities of proteins for characterization is paramount to the success or failure of the project. Previous studies with piggyBac systems have underscored the ability to increase the titer of recombinant proteins in cell pools by five-fold with a concomitant increase in specific productivity and expression stability in excess of 16 weeks.
Small, preclinical-scale batches are typically made utilizing transient transfection of large quantities of expression plasmid, to which selection may or may not be employed to enrich for higher expressing clones within the pool. Recent studies from the Biotechnology Discovery Research group at Eli Lilly have confirmed the use of the PB system to increase integration rates, leading to significant improvements in the titers generated from these pools.
Figure 2 shows the improvement in titer for four different antibodies from pools generated using piggyBac relative to conventional methods. Specific productivity rates in the pools were as high as those seen from clonal manufacturing lines. This scalable method was able to generate the required levels of protein in only a couple of weeks.
Figure 2. Expression levels of pools of cells for four different recombinant antibodies, using standard transfection or integration with piggyBac. Adapted from Rajendra et al., Biotechnology Progress, 2016.
Once a final molecule has been chosen for the candidate therapeutic, it is best to maximize reproducibility of the manufacturing cell line by isolating a single high-expressing clone. In this case, banked versions of the previously validated piggyBac pools can be used to identify these clones, with a significant time and titer advantage over traditional stable cell line generation.
Figure 3 shows the volumetric titer of clones isolated from these pools to be much higher than the control system, with only 12% of the piggyBac clones categorized as low producers (
When further segmented, the top 24 clones produced with piggyBac were all higher than those generated using traditional methods. Given the high likelihood of finding a high-producing clone in the piggyBac™ pools, the amount of screening required to move forward is greatly decreased.
Figure 3. (A) Number of clones rated as high (>20 mg/L), medium (10-20 mg/L), or low expressers (
Hard to Deliver Projects
In addition to the increased titer and shortened timelines for the expression of traditional proteins, piggyBac offers advantages with regard to delivery of nonstandard proteins. It is not uncommon for projects to be terminated solely due to the inability to reliably generate sufficient quantities of the recombinant protein. Reasons for inconsistent expression can include a requirement for multi-subunit proteins, exceedingly large proteins, or any other protein that is simply inefficient to make, such as bi-specific antibodies.
Expression of multiple recombinant proteins in one cell line could be required for either the production of multi-subunit proteins or for the inclusion of additional effector proteins necessary to enhance expression or functionality. The PB system has been used repeatedly to efficiently express multiple proteins in one cell, whether as a protein complex or as separate proteins. This highlights another aspect of the PB system, the ability to integrate large cargoes (≥200 Kb in size), which allows the efficient expression of large proteins that are not possible using other integration methods.
These two additional aspects, along with the ability to skew toward transcriptionally active integration sites, makes the PB system ideal for the recovery of projects that were previously not possible. The capability to resurrect shelved drug discovery projects that might have been good candidates, but could not be produced at scale is important for further biotherapeutic production workflows.
Summary
As biopharma continues to evolve, drug manufacturers face an array of challenges related to efficiency and productivity. From better expression systems to purification improvements and process stabilization, recent research into new ways to reliably produce recombinant protein in large quantities has identified the piggyBac transposon system as a potential key to alleviating this frustrating bottleneck.
Production of sufficient levels of therapeutic proteins, even in the preclinical arena, in a time and cost-effective manner, helps to quickly make the go/no-go decisions required to continue development or fail quickly.
