Sustaining an efficient and effective CHO cell line development platform by incorporation of 24-deep well plate screening and multivariate analysis.

Efficient and effective cell line screening is paramount toward a successful biomanufacturing program. Here we describe the implementation of 24-deep well plate (24-DWP) screening of CHO lines as part of the cell line development platform at AbbVie. Incorporation of this approach accelerated the identification of the best candidate lines for process development. In an effort to quantify and predict process performance comparability, we compared cell culture performance in and in shake flasks, for a panel of Chinese Hamster Ovary cell lines expressing a monoclonal antibody. The results in 24-DWP screening showed reduced growth profiles, but comparable viability profiles. Slow growers in 24-DWP achieved the highest productivity improvement upon scaling-up to shake flasks. Product quality of the protein purified from shake flasks and 24-DWP were also compared. The 24-DWP culture conditions were found to influence the levels of acidic species, reduce the G0 N-glycan species, and increase the high-mannose N-glycan species. Nevertheless, the identification of undesirable profiles is executed consistently with the scaled-up culture. We further employed multivariate data analysis to capture differences depending on the two scales and we could demonstrate that cell line profiles were adequately clustered, regardless of the vessel used for the development. In conclusion, the 24-DWP platform was reasonably predictive of the parameters crucial for upstream process development activities, and has been adapted as part of the AbbVie cell line development platform. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:175-186, 2018.

Arabinosylation of recombinant human immunoglobulin-based protein therapeutics.

Protein glycosylation is arguably the paramount post-translational modification on recombinant glycoproteins, and highly cited in the literature for affecting the physiochemical properties and the efficacy of recombinant glycoprotein therapeutics. Glycosylation of human immunoglobulins follows a reasonably well-understood metabolic pathway, which gives rise to a diverse range of asparagine-linked (N-linked), or serine/threonine-linked (O-linked) glycans. In N-linked glycans, fucose levels have been shown to have an inverse relationship with the degree of antibody-dependent cell-mediated cytotoxicity, and high mannose levels have been implicated in potentially increasing immunogenicity and contributing to less favorable pharmacokinetic profiles. Here, we demonstrate a novel approach to potentially reduce the presence of high-mannose species in recombinant human immunoglobulin preparations, as well as facilitate an approximate 100% replacement of fucosylation with arabinosylation in Chinese hamster ovary cell culture through media supplementation with D-arabinose, an uncommonly used mammalian cell culture sugar substrate. The replacement of fucose with arabinose was very effective and practical to implement, since no cell line engineering or cellular adaptation strategies were required. Arabinosylated recombinant IgGs and the accompanying reduction in high mannose glycans, facilitated a reduction in dendritic cell uptake, increased FcγRIIIa signaling, and significantly increased the levels of ADCC. These aforementioned effects were without any adverse changes to various structural or functional attributes of multiple recombinant human antibodies and a bispecific DVD-Ig. Protein arabinosylation represents an expansion of the N-glycan code in mammalian expressed glycoproteins.

Cell culture media supplementation of infrequently used sugars for the targeted shifting of protein glycosylation profiles.

Mammalian cells in culture rely on sources of carbohydrates to supply the energy requirements for proliferation. In addition, carbohydrates provide a large source of the carbon supply for supporting various other metabolic activities, including the intermediates involved in the protein glycosylation pathway. Glucose and galactose, in particular, are commonly used sugars in culture media for these purposes. However, there exists a very large repertoire of other sugars in nature, and many that have been chemically synthesized. These sugars are particularly interesting because they can be utilized by cells in culture in distinct ways. In the present work it has been found that many infrequently used sugars, and the corresponding cellular response towards them as substrates, led to differences in the protein N-glycosylation profile of a recombinant glycoprotein. The selective media supplementation of raffinose, trehalose, turanose, palatinose, melezitose, psicose, lactose, lactulose, and mannose were found to be capable of redirecting N-glycan oligosaccharide profiles. Despite this shifting of protein glycosylation, there were no other adverse changes in culture performance, including both cell growth and cellular productivity over a wide range of supplemented sugar concentrations. The approach presented highlights a potential means towards both the targeted shifting of protein glycosylation profiles and ensuring recombinant protein comparability, which up to this point in time has remained under-appreciated for these under-utilized compounds. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:511-522, 2017.

When Good Intentions Go Awry: Modification of a Recombinant Monoclonal Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbic Acid.

With the advent of new initiatives to develop chemically defined media, cell culture scientists screen many additives to improve cell growth and productivity. However, the introduction or increase of supplements, typically considered beneficial or protective on their own, to the basal media or feed stream may cause unexpected detrimental consequences to product quality. For instance, because cultured cells are constantly under oxidative stress, ascorbic acid (vitamin C, a potent natural reducing agent) is a common additive to cell culture media. However, as reported herein, a recombinant monoclonal antibody (adalimumab) in cell culture was covalently modified by xylosone (molecular weight 148), an oxidative product of ascorbate. Containing reactive carbonyl groups, xylosone modifies various amines (e.g., the N-termini of the heavy and light chains and susceptible lysines), forming either hemiaminal (+148 Da) or Schiff base (imine, +130 Da) products. Our findings show, for the first time, that ascorbate-derived xylosone can contribute to an increase in molecular heterogeneity, such as acidic species. Our work serves as a reminder that additives to cell culture and their metabolites may become reactive and negatively impact the overall product quality and should be carefully monitored with any changes in cell culture conditions.

Cell culture media supplementation of bioflavonoids for the targeted reduction of acidic species charge variants on recombinant therapeutic proteins.

Charge variants in recombinant proteins are an important series of protein modifications, whose potential role on protein stability, activity, immunogenicity, and pharmacokinetics continues to be studied. Monoclonal antibodies in particular have been shown to have a wide range of acidic species variants, including those associated with the addition of covalent modifications as well as the chemical degradation at specific peptide regions on the antibody. These variants play a significant role toward the overall heterogeneity of recombinant therapeutic proteins and are typically monitored during manufacturing to ensure they lie within proven acceptable ranges. In this work, it has been found that the supplementation of members of the bioflavonoid chemical family into mammalian cell culture media was effective toward the reduction of acidic species charge variants on recombinant monoclonal antibodies and dual variable domain immunoglobulins. The demonstrated reduction in acidic species through the use of bioflavonoids facilitates the manufacturing of a less heterogeneous product with potential improvements in antibody structure and function.

Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics.

Protein glycosylation is an important post-translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N-glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody-dependent cell-mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability.

Improvement of mammalian cell culture performance through surfactant enabled concentrated feed media.

The design of basal and feed media in mammalian cell culture is paramount towards ensuring acceptable upstream process performance in various operation modes, especially fed-batch culture. Mammalian cell culture media designs have evolved from the classical formulations designed by Eagle and Ham, to today's formulations designed from continuous improvement and statistical frameworks. Feed media is especially important for ensuring robust cell growth, productivity, and ensuring the product quality of recombinant therapeutics are within acceptable ranges. Numerous studies have highlighted the benefit of various media designs, supplements, and feed addition strategies towards the resulting cell culture process. In this work we highlight the use of a top-down level approach towards feed media design enabled by the use of select surfactants for the targeted enrichment of a chemically defined feed media. The use of the enriched media was able to improve product titers at g/L levels, without adversely impacting the growth of multiple Chinese Hamster Ovary cell lines or the product quality of multiple recombinant antibodies.

Protein glycosylation control in mammalian cell culture: past precedents and contemporary prospects.

Protein glycosylation is a post-translational modification of paramount importance for the function, immunogenicity, and efficacy of recombinant glycoprotein therapeutics. Within the repertoire of post-translational modifications, glycosylation stands out as having the most significant proven role towards affecting pharmacokinetics and protein physiochemical characteristics. In mammalian cell culture, the understanding and controllability of the glycosylation metabolic pathway has achieved numerous successes. However, there is still much that we do not know about the regulation of the pathway. One of the frequent conclusions regarding protein glycosylation control is that it needs to be studied on a case-by-case basis since there are often conflicting results with respect to a control variable and the resulting glycosylation. In attempts to obtain a more multivariate interpretation of these potentially controlling variables, gene expression analysis and systems biology have been used to study protein glycosylation in mammalian cell culture. Gene expression analysis has provided information on how glycosylation pathway genes both respond to culture environmental cues, and potentially facilitate changes in the final glycoform profile. Systems biology has allowed researchers to model the pathway as well-defined, inter-connected systems, allowing for the in silico testing of pathway parameters that would be difficult to test experimentally. Both approaches have facilitated a macroscopic and microscopic perspective on protein glycosylation control. These tools have and will continue to enhance our understanding and capability of producing optimal glycoform profiles on a consistent basis.

Optimal and consistent protein glycosylation in mammalian cell culture.

In the biopharmaceutical industry, mammalian cell culture systems, especially Chinese hamster ovary (CHO) cells, are predominantly used for the production of therapeutic glycoproteins. Glycosylation is a critical protein quality attribute that can modulate the efficacy of a commercial therapeutic glycoprotein. Obtaining a consistent glycoform profile in production is desired due to regulatory concerns because a molecule can be defined by its carbohydrate structures. An optimal profile may involve a spectrum of product glycans that confers a desired therapeutic efficacy, or a homogeneous glycoform profile that can be systemically screened for. Studies have shown some degree of protein glycosylation control in mammalian cell culture, through cellular, media, and process effects. Studies upon our own bioprocesses to produce fusion proteins and monoclonal antibodies have shown an intricate relationship between these variables and the resulting protein quality. Glycosylation optimization will improve therapeutic efficacy and is an ongoing goal for researchers in academia and industry alike. This review will focus on the advancements made in glycosylation control in a manufacturing process, as well as the next steps in understanding and controlling protein glycosylation.

Systems analysis of N-glycan processing in mammalian cells.

N-glycosylation plays a key role in the quality of many therapeutic glycoprotein biologics. The biosynthesis reactions of these oligosaccharides are a type of network in which a relatively small number of enzymes give rise to a large number of N-glycans as the reaction intermediates and terminal products. Multiple glycans appear on the glycoprotein molecules and give rise to a heterogeneous product. Controlling the glycan distribution is critical to the quality control of the product. Understanding N-glycan biosynthesis and the etiology of microheterogeneity would provide physiological insights, and facilitate cellular engineering to enhance glycoprotein quality. We developed a mathematical model of glycan biosynthesis in the Golgi and analyzed the various reaction variables on the resulting glycan distribution. The Golgi model was modeled as four compartments in series. The mechanism of protein transport across the Golgi is still controversial. From the viewpoint of their holding time distribution characteristics, the two main hypothesized mechanisms, vesicular transport and Golgi maturation models, resemble four continuous mixing-tanks (4CSTR) and four plug-flow reactors (4PFR) in series, respectively. The two hypotheses were modeled accordingly and compared. The intrinsic reaction kinetics were first evaluated using a batch (or single PFR) reactor. A sufficient holding time is needed to produce terminally-processed glycans. Altering enzyme concentrations has a complex effect on the final glycan distribution, as the changes often affect many reaction steps in the network. Comparison of the glycan profiles predicted by the 4CSTR and 4PFR models points to the 4PFR system as more likely to be the true mechanism. To assess whether glycan heterogeneity can be eliminated in the biosynthesis of biotherapeutics the 4PFR model was further used to assess whether a homogeneous glycan profile can be created through metabolic engineering. We demonstrate by the spatial localization of enzymes to specific compartments all terminally processed N-glycans can be synthesized as homogeneous products with a sufficient holding time in the Golgi compartments. The model developed may serve as a guide to future engineering of glycoproteins.

Engineering cells for cell culture bioprocessing--physiological fundamentals.

In the past decade, we have witnessed a tremendous increase in the number of mammalian cell-derived therapeutic proteins with clinical applications. The success of making these life-saving biologics available to the public is partly due to engineering efforts to enhance process efficiency. To further improve productivity, much effort has been devoted to developing metabolically engineered producing cells, which possess characteristics favorable for large-scale bioprocessing. In this article we discuss the fundamental physiological basis for cell engineering. Different facets of cellular mechanisms, including metabolism, protein processing, and the balancing pathways of cell growth and apoptosis, contribute to the complex traits of favorable growth and production characteristics. We present our assessment of the current state of the art by surveying efforts that have already been undertaken in engineering cells for a more robust process. The concept of physiological homeostasis as a key determinant and its implications on cell engineering is emphasized. Integrating the physiological perspective with cell culture engineering will facilitate attainment of dream cells with superlative characteristics.

GlycoVis: visualizing glycan distribution in the protein N-glycosylation pathway in mammalian cells.

Glycosylation has profound effects on the quality of recombinant proteins produced in mammalian cells. The biosynthetic pathways of N-linked glycans on glycoproteins involves a relatively small number of enzymes and nucleotide sugars. Many of these glycoconjugate enzymes can utilize multiple N-glycans as substrates, thus generating a large number of glycan intermediates, and making the biosynthetic pathway resemble a network with diverging and converging paths. The N-glycans on secreted glycoprotein molecules include not only terminal glycans, but also pathway intermediates. To better assess the glycan distribution and the potential route of their synthesis, we created GlycoVis, a visualization program that displays the distribution and the potential reaction paths leading to each N-glycan on the reaction network. The substrate specificities of the enzymes involved were organized into a relationship matrix. With the input of glycan distribution data, the program outputs a reaction pathway map which labels the relative abundance levels of different glycans with different colors. The program also traces all possible reaction paths leading to each glycan and identifies each pathway on the map. Glycoform distribution of Chinese Hamster Ovary cell-derived tissue plasminogen activator (TPA), and human and mouse IgG were used as illustrations for the application of GlycoVis. In addition, the intracellular and secreted IgG from an NS0 producer cell line were isolated, and their glycoform profiles were displayed using GlycoVis for comparison. This visualization tool facilitates the analysis of potential reaction paths utilized under different physiological or culture conditions, and may provide insight on the potential targets for metabolic engineering.