The manufacturing of recombinant protein is traditionally divided in two main steps: upstream (cell culture and synthesis of the target protein) and downstream (purification and formulation of the protein into a drug substance or drug product). Today, cost pressure, market uncertainty and market growth, challenge the existing manufacturing technologies. Leaders in the field are active in designing the process of the future and continuous manufacturing is recurrently mentioned as a potential solution to address some of the current limitations. This review focuses on the application of continuous processing to the first step of the manufacturing process. Enabling technologies and operation modes are described in the first part. In the second part, recent advances in the field that have the potential to support its successful future development are critically discussed.

No abstract

Quality by Design (QbD) is a new approach to the development of recombinant therapeutic protein products that promotes a better understanding of the product and its manufacturing process. The first step in the QbD approach consists in identifying the critical quality attributes (CQA), i.e., those quality attributes of the product that have an impact on its clinical efficacy or safety. CQAs are identified through a science-based risk assessment taking into consideration a combination of clinical and nonclinical data obtained with the molecule or other similar molecules or platform products, as well as the published literature. The purpose of this article is to perform a comprehensive review of the published literature, supporting an assessment of the impact on safety and efficacy of the quality attributes commonly encountered in recombinant therapeutic proteins, more specifically those produced in mammalian cell expression systems. Quality attributes generally observed in biopharmaceutical proteins including product-related impurities and substances, process-related impurities, product attributes, and contaminants are evaluated one by one for their impact on biological activity, pharmacokinetics and pharmacodynamics, immunogenicity, and overall safety/toxicity.

A host-cell-related proteolytic activity was identified in a recombinant Fc-fusion protein production process. This report describes the strategy applied to characterize and isolate the enzyme responsible for this degradation by combining cell culture investigation and dedicated analytical tools. After isolation and sequencing of the clipped fragment generated in post-capture material, enzymatic activity was traced in different culture conditions, allowing identification of viable CHO cells as the source of protease. Inhibitors and pH screenings showed that the enzyme belongs to an aspartic protease family and is preferably active at acidic pH. The protease was isolated by purification on a pepstatin A column and characterized as a protein related to cathepsin D. An additional metallo-protease inhibited by EDTA was identified with an optimum activity at neutral pH. This study is an example of how quality and stability of therapeutic recombinant molecules are strongly influenced by cell culture parameters.

Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can conclude that the modulation of glycosylation in a sequential steady state approach in combination with mechanistic model represents an efficient and rational strategy to develop continuous processes with desired N-linked glycosylation patterns. Biotechnol. Bioeng. 2017;114: 1978-1990. © 2017 Wiley Periodicals, Inc.