Process development and characterization for integrated continuous bioprocesses-Highlights from N-mAb.

The N-mAb case study was produced by the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) to support teaching and learning for both industry and to accelerate adoption of advanced manufacturing process technologies such as integrated continuous bioprocesses (ICB) for mAbs. Similar to the A-mAb case study, N-mAb presents the evolution of an integrated control strategy, from early clinical through process validation and commercial manufacturing with a focus on elements that are unique to integrated continuous bioprocesses. This publication presents a summary of the process design and characterization chapters to allow a greater focus on the unique elements relevant to that phase of development.

A rapid method for relative quantification of N-glycans from a therapeutic monoclonal antibody during trastuzumab biosimilar development.

Glycosylation is a common post-translational modification and critical quality attribute that can modulate the efficacy of therapeutic proteins. In the production of monoclonal antibodies (mAbs), quantifying the glycoform profile is a vital characterization step. Traditional glycan analysis is time consuming and involves steps at extreme temperature or pH, which may alter glycans. Here, we describe a rapid method for glycan analysis in which glycans are released from mAb samples that are bound to protein A columns. Since host cell proteins, which may also contain glycans, were already removed, this step enables analysis of cell culture products. Glycans released from the mAb samples are then derivatized with InstantPC™ labeling agent and analyzed by HILIC-FLD-MS. To illustrate the method, the glycan profiles of six trastuzumab (Herceptin®) antibody lots and four biosimilar developmental lots were analyzed. The results derived from our novel method, which takes less than 90 min, are compared with those from a typical glycan preparation approach.

The potential of hydrodynamic damage to animal cells of industrial relevance: current understanding.

Suspension animal cell culture is now routinely scaled up to bioreactors on the order of 10,000 L, and greater, to meet commercial demand. However, the concern of the 'shear sensitivity' of animal cells still remains, not only within the bioreactor, but also in the downstream processing. As the productivities continue to increase, titer of ~10 g/L are now reported with cell densities greater than 2 × 10(7) cells/mL. Such high, and potentially higher cell densities will inevitably translate to increased demand in mass transfer and mixing. In addition, achieving productivity gains in both the upstream stage and downstream processes can subject the cells to aggressive environments such as those involving hydrodynamic stresses. The perception of 'shear sensitivity' has historically put an arbitrary upper limit on agitation and aeration in bioreactor operation; however, as cell densities and productivities continue to increase, mass transfer requirements can exceed those imposed by these arbitrary low limits. Therefore, a better understanding of how animal cells, used to produce therapeutic products, respond to hydrodynamic forces in both qualitative and quantitative ways will allow an experimentally based, higher, "upper limit" to be created to guide the design and operation of future commercial, large scale bioreactors. With respect to downstream hydrodynamic conditions, situations have already been achieved in which practical limits with respect to hydrodynamic forces have been experienced. This review mainly focuses on publications from both the academy and industry regarding the effect of hydrodynamic forces on industrially relevant animal cells, and not on the actual scale-up of bioreactors. A summary of implications and remaining challenges will also be presented.

Computer simulations of the energy dissipation rate in a fluorescence-activated cell sorter: Implications to cells.

Fluorescence activated cell sorting, FACS, is a widely used method to sort subpopulations of cells to high purities. To achieve relatively high sorting speeds, FACS instruments operate by forcing suspended cells to flow in a single file line through a laser(s) beam(s). Subsequently, this flow stream breaks up into individual drops which can be charged and deflected into multiple collection streams. Previous work by Ma et al. (2002) and Mollet et al. (2007; Biotechnol Bioeng 98:772-788) indicates that subjecting cells to hydrodynamic forces consisting of both high extensional and shear components in micro-channels results in significant cell damage. Using the fluid dynamics software FLUENT, computer simulations of typical fluid flow through the nozzle of a BD FACSVantage indicate that hydrodynamic forces, quantified using the scalar parameter energy dissipation rate, are similar in the FACS nozzle to levels reported to create significant cell damage in micro-channels. Experimental studies in the FACSVantage, operated under the same conditions as the simulations confirmed significant cell damage in two cell lines, Chinese Hamster Ovary cells (CHO) and THP1, a human acute monocytic leukemia cell line.

Acute hydrodynamic forces and apoptosis: a complex question.

A second generation flow contraction device was developed and modeled which allows cells to be subjected to well-defined hydrodynamic forces. Studies were conducted with this system on wild-type Chinese Hamster Ovary cells (CHO-K1) and a strain of CHO cells which expresses the human Bcl-2 triangle gene (CHO-bcl-2). In this study, the following questions were asked: (1) Does an acute hydrodynamic force induce apoptosis in wild-type CHO and CHO-bcl-2 cells? (2) Does the type of culture media make a difference with respect to the induction of apoptosis or necrosis? and (3) Does culture history affect induction of apoptosis or necrosis? The results obtained with this new flow contraction device and corresponding computer simulations are consistent with previously published studies with respect to the level of energy dissipation rate (EDR) required to create significant cell lysis. Second, while detectable relative to the control in the T-flask experiments, only a small fraction of the cells become apoptotic when exposed to a sub-lysis level of EDR (<10(8) W x m(-3)). Third, cells cultured in suspension with serum free media do not exhibit any higher or lower sensitivity (with respect to apoptosis) to various levels of EDR when compared to control cultures grown in T-flask and serum containing media; on the other hand, necrosis is significantly increased in experiments performed on suspended cells without serum. Fourth, the addition of the Bcl-2 gene product might slightly reduce the occurrence of apoptosis in T-flask culture; however, the baseline response is so low that the difference is insignificant.