A common framework for integrated and continuous biomanufacturing.

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.

Modeling the Effect of Amino Acids and Copper on Monoclonal Antibody Productivity and Glycosylation: A Modular Approach.

In manufacturing monoclonal antibodies (mAbs), it is crucial to be able to predict how process conditions and supplements affect productivity and quality attributes, especially glycosylation. Supplemental inputs, such as amino acids and trace metals in the media, are reported to affect cell metabolism and glycosylation; quantifying their effects is essential for effective process development. We aim to present and validate, through a commercially relevant cell culture process, a technique for modeling such effects efficiently. While existing models can predict mAb production or glycosylation dynamics under specific process configurations, adapting them to new processes remains challenging, because it involves modifying the model structure and often requires some mechanistic understanding. Here, a modular modeling technique for adapting an existing model for a fed-batch Chinese hamster ovary (CHO) cell culture process without structural modifications or mechanistic insight is presented. Instead, data is used, obtained from designed experimental perturbations in media supplementation, to train and validate a supplemental input effect model, which is used to "patch" the existing model. The combined model can be used for model-based process development to improve productivity and to meet product quality targets more efficiently. The methodology and analysis are generally applicable to other CHO cell lines and cell types.

Raman spectroscopic based chemometric models to support a dynamic capacitance based cell culture feeding strategy.

Multiple process analytical technology (PAT) tools are now being applied in tandem for cell culture. Research presented used two in-line probes, capacitance for a dynamic feeding strategy and Raman spectroscopy for real-time monitoring. Data collected from eight batches at the 15,000 L scale were used to develop process models. Raman spectroscopic data were modelled using Partial Least Squares (PLS) by two methods-(1) use of the full dataset and (2) split the dataset based on the capacitance feeding strategy. Root mean square error of prediction (RMSEP) for the first model method of capacitance was 1.54 pf/cm and the second modelling method was 1.40 pf/cm. The second Raman method demonstrated results within expected process limits for capacitance and a 0.01% difference in total nutrient feed compared to the capacitance probe. Additional variables modelled using Raman spectroscopy were viable cell density (VCD), viability, average cell diameter, and viable cell volume (VCV).

Reduced scale model qualification of 5-L and 250-ml bioreactors using multivariant visualization and Bayesian inferential methods.

A novel method for the qualification of reduced scale models (RSMs) was illustrated using data from both a 250-ml advanced microscale bioreactor (ambr) and a 5-L bioreactor RSM for a 2,000-L manufacturing scale process using a CHO cell line to produce a recombinant monoclonal antibody. The example study showed how the method was used to identify process performance attributes and product quality attributes that capture important aspects of the RSM qualification process. The method uses two novel statistical approaches: multivariate dimension reduction and data visualization techniques, via partial least squares discriminant analysis (PLS-DA), and Bayesian multivariate linear modeling for inferential analysis. Bayesian multivariate linear modeling allows for individual probability distributions of the differences of the mean of each attribute for each scale, as well as joint probability statements on the differences of the means for multiple attributes. Depending on the results of this inferential procedure, PLS-DA is used to identify the process performance outputs at the different scales which have the greatest negative impact on the multivariate Bayesian joint probabilities. Experience with that particular process can then be leveraged to adjust operating conditions to minimize these differences, and then equivalence can be reassessed using the multivariate linear model.

Optimizing performance of semi-continuous cell culture in an ambr15™ microbioreactor using dynamic flux balance modeling.

The ambr bioreactors are single-use microbioreactors for cell line development and process optimization. With operating conditions for large-scale biopharmaceutical production properly scaled down, microbioreactors such as the ambr15™ can potentially be used to predict the effect of process changes such as modified media or different cell lines. While there have been some recent studies evaluating the ambr15™ technology as a scale-down model for fed-batch operations, little has been reported for semi-continuous or continuous operation. Gassing rates and dilution rates in the ambr15™ were varied in this study to attempt to replicate performance of a perfusion process at the 5 L scale. At both scales, changes to metabolite production and consumption, and cell growth rate and therapeutic protein production were measured. Conditions were identified in the ambr15™ bioreactor that produced metabolic shifts and specific metabolic and protein production rates that are characteristic of the corresponding 5 L perfusion process. A dynamic flux balance (DFB) model was employed to understand and predict the metabolic changes observed. The DFB model predicted trends observed experimentally, including lower specific glucose consumption and a switch from lactate production to consumption when dissolved CO2 was maintained at higher levels in the broth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:420-431, 2018.

Microarray platform affords improved product analysis in mammalian cell growth studies.

High throughput (HT) platforms serve as a cost-efficient and rapid screening method for evaluating the effect of cell-culture conditions and screening of chemicals. We report the development of a HT cell-based microarray platform to assess the effect of culture conditions on Chinese hamster ovary (CHO) cells. Specifically, growth, transgene expression and metabolism of a GS/methionine sulphoximine (MSX) CHO cell line, which produces a therapeutic monoclonal antibody, was examined using a microarray system in conjunction with a conventional shake flask platform in a non-proprietary medium. The microarray system consists of 60-nL spots of cells encapsulated in alginate and separated in groups via an 8-well chamber system attached to the chip. Results show the non-proprietary medium developed allows cell growth, production, and normal glycosylation of recombinant antibody and metabolism of the recombinant CHO cells in both the microarray and shake flask platforms. In addition, 10.3 mM glutamate addition to the defined base medium results in lactate metabolism shift in the recombinant GS/MSX CHO cells in the shake flask platform. Ultimately, the results demonstrate that the HT microarray platform has the potential to be utilized for evaluating the impact of media additives on cellular processes, such as cell growth, metabolism, and productivity.

Development of a Chinese hamster ovary cell line for recombinant adenovirus-mediated gene expression.

Recombinant human adenovirus (rhAd) has been used extensively for functional protein expression in mammalian cells including those of human and nonhuman origin. High-level protein production by rhAd vectors is expected in their permissive host cells, such as the human embryonic kidney 293 (HEK293) cell line. This is attributed primarily to the permissiveness of HEK293 to rhAd infection and their ability to support viral DNA replication by providing the missing El proteins. However, the HEK293 cells tend to suffer from cytopathic effect (CPE) as a result of virus replication. Under these circumstances, the host cell function is compromised and the culture viability will be reduced. Consequently, newly synthesized polypeptides may not be processed properly at posttranslational levels. Therefore, the usefulness of HEK293 cells for the expression of complex targets such as secreted proteins could be limited. In the search for a more robust cell line as a production host for rhAd expression vectors, a series of screening experiments was performed to isolate clones from Chinese hamster ovary-K1 (CHO-K1) cells. First, multiple rounds of infection of CHO-K1 cells were performed utilizing an rhAd expressing GFP. After each cycle of infection, a small population of CHO cells with high GFP levels was enriched by FACS. Second, individual clones more permissive to human adenovirus infection were isolated from the highly enriched subpopulation by serial dilution. A single clone, designated CHO-K1-C5, was found to be particularly permissive to rhAd infection than the parental pool and has served as a production host in the successful expression of several secreted proteins.