Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of biopharmaceutical products has grown significantly so that, as of November 10, 2014, forty-seven monoclonal antibody products have been approved in the US or Europe for the treatment of a variety of diseases, and many of these products have also been approved for other global markets. At the current approval rate of ∼ four new products per year, ∼ 70 monoclonal antibody products will be on the market by 2020, and combined world-wide sales will be nearly $125 billion.

https://www.tandfonline.com/doi/pdf/10.4161/19420862.2015.989042

The therapeutic monoclonal antibody market

Low oxygen tension is thought to be an integral component of the human mesenchymal stem cell (hMSC) native bone marrow microenvironment. HMSC were cultured under physiologically relevant oxygen environments (2% O2) in three-dimensional (3D) constructs for up to 1 month in order to investigate the combined effects of chronic hypoxia and 3D architecture on hMSC tissue-development patterns. Hypoxic hMSC exhibited an extended lag phase in order to acclimatize to culture conditions. However, they subsequently proliferated continuously throughout the culture period, while maintaining significantly higher colony-forming unit capabilities and expressing higher levels of stem cell genes than hMSC cultured at 20% O2 (normoxic) conditions. Upon induction, hypoxic hMSC also expressed higher levels of osteoblastic and adipocytic differentiation markers than normoxic controls. Hypoxia induced increased total protein levels in hMSC throughout the culture period, as well as significantly different fibronectin expression patterns suggesting that oxygen levels can significantly affect tissue-development patterns. Importantly, hMSC maintained the ability to thrive in prolonged hypoxic conditions suggesting that hypoxia may be an essential element of the in vivo hMSC niche. Further studies are required to determine how variations in cellular characteristics and ECM expression impact on the physiological properties of the engineered tissue, yet these results strongly indicate that oxygen tension is a key parameter that influences the in vitro characteristics of hMSC and their development into tissues.

Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs

This article mainly addresses the issues associated with the engineering of large-scale free suspension culture in agitated bioreactors >10,000 L because they have become the system of choice industrially. It is particularly concerned with problems that become increasingly important as the scale increases. However, very few papers have been written that are actually based on such large-scale studies and the few that do rarely address any of the issues quantitatively. Hence, it is necessary very often to extrapolate from small-scale work and this review tries to pull the two types of study together. It is shown that 'shear sensitivity' due to agitation and bursting bubbles is no longer considered a major problem. Homogeneity becomes increasingly important with respect to pH and nutrients at the largest scale and sub-surface feeding is recommended despite 'cleaning in place' concerns. There are still major questions with cell retention/recycle systems at these scales, either because of fouling, of capacity or of potential and different 'shear sensitivity' questions. Fed-batch operation gives rise to cell densities that have led to the use of oxygen and enriched air to meet oxygen demands. This strategy, in turn, gives rise to a CO(2) evolution rate that impacts on pH control, pCO(2) and osmolality. These interactions are difficult to resolve but if higher sparge and agitation intensities could be used to achieve the necessary oxygen transfer, the problem would largely disappear. Thus, the perception of 'shear sensitivity' is still impacting on the development of animal cell culture at the commercial scale. Microcarrier culture is also briefly addressed. Finally, some recommendations for bioreactor configuration and operating strategy are given.

Reactor engineering in large scale animal cell culture.

Although O(2) concentrations are considerably lowered in vivo, depending on the tissue and cell population in question (some cells need almost anoxic environment for their maintenance) the cell and tissue cultures are usually performed at atmospheric O(2) concentration (20-21%). As an instructive example, the relationship between stem cells and micro-environmental/culture oxygenation has been recapitulated. The basic principle of stem cell biology, "the generation-age hypothesis," and hypoxic metabolic properties of stem cells are considered in the context of the oxygen-dependent evolution of life and its transposition to ontogenesis and development. A hypothesis relating the self-renewal with the anaerobic and hypoxic metabolic properties of stem cells and the actual O(2) availability is elaborated ("oxygen stem cell paradigm"). Many examples demonstrated that the cellular response is substantially different at atmospheric O(2) concentration when compared to lower O(2) concentrations which better approximate the physiologic situation. These lower O(2) concentrations, traditionally called "hypoxia" represent, in fact, an in situ normoxia, and should be used in experimentation to get an insight of the real cell/cytokine physiology. The revision of our knowledge on cell/cytokine physiology, which has been acquired ex vivo at non physiological atmospheric (20-21%) O(2) concentrations representing a hyperoxic state for most primate cells, has thus become imperious.

Hypoxia or in situ normoxia: The stem cell paradigm

The presence of spatial gradients in fundamental culture parameters, such as dissolved gases, pH, concentration of substrates, and shear rate, among others, is an important problem that frequently occurs in large-scale bioreactors. This problem is caused by a deficient mixing that results from limitations inherent to traditional scale-up methods and practical constraints during large-scale bioreactor design and operation. When cultured in a heterogeneous environment, cells are continuously exposed to fluctuating conditions as they travel through the various zones of a bioreactor. Such fluctuations can affect cell metabolism, yields, and quality of the products of interest. In this review, the theoretical analyses that predict the existence of environmental gradients in bioreactors and their experimental confirmation are reviewed. The origins of gradients in common culture parameters and their effects on various organisms of biotechnological importance are discussed. In particular, studies based on the scale-down methodology, a convenient tool for assessing the effect of environmental heterogene ities, are surveyed.

Living with heterogeneities in bioreactors: understanding the effects of environmental gradients on cells.

Megakaryocytes (Mks) mature adjacent to bone marrow (BM) sinus walls and subsequently release platelets within the sinusoidal space or in lung capillaries. As the sites for platelet release have higher levels of oxygen tension (pO2) than the core of the BM where stem and progenitor cells reside, we investigated whether pO2 influences Mk maturation. Mks were generated from CD34+ cells (from mobilized peripheral blood from cancer patients) under 5% and 20% O2. At day 15, CD41+ Mk expansion in 20% and 5% O2 cultures was 85-fold and 31-fold respectively. Twenty percent O2 cultures also had higher levels of high ploidy (≥ 8N, eightfold higher) and proplatelet-forming (fivefold higher) Mks. At day 21, 20% O2 cultures had a fivefold higher number of apoptotic Mks. In contrast, 5% O2 promoted Mk colony-forming unit (CFU-Mk) generation and maintenance. Similar results were observed in cultures initiated with CD41+ Mks, indicating that pO2 directly affects Mks. The change from 20% to 5% O2 on day 5 and day 7 delayed both maturation and apoptosis, suggesting that these two processes are closely linked. These results were confirmed in CD34+ cultures from normal BM samples. These data may provide insights into in vivo Mk maturation, such as an explanation for hypoxia-induced thrombocytopenia in animals.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2141.2000.02457.x

Oxygen tension influences the differentiation, maturation and apoptosis of human megakaryocytes

Carbon dioxide buildup in large-scale reactors can be detrimental to cell growth and productivity. In case of protein X, a therapeutic glycoprotein, when cultures were scaled up from bench scale to the pilot plant, there was a 40% loss of specific productivity. The dissolved CO(2) (dCO(2)) level was 179 +/- 9 mmHg at the pilot plant scale and 68 +/- 13 mmHg at bench scale. The authors proposed a comprehensive approach to maintain dCO(2) levels between 40 and 120 mmHg throughout the 14-day fed-batch process. A cell-free experiment was used to investigate the impact of the following parameters on dCO(2) removal: (1) sparge rate, (2) agitator speed, (3) bubble size, (4) bicarbonate concentration, (5) impeller position, and (6) aeration rate at the headspace of bioreactor. dCO(2) was measured using a fiber optic based probe. dCO(2) removal rate was a strong function of sparge rate and a weak function of agitator speed. Bubble size was modulated by the presence or absence of a sparge stone (10 microm pore size, 1 cm pipe i.d.). Open pipe provided 3- to 4-fold better dCO(2) removal for the same mass transfer coefficient (k(L)a) value. A mathematical model and a bench-scale experiment indicated that the benefit of a lower level of sodium bicarbonate in the culture medium was transient for batch and fed-batch cultures. Thus, this strategy was not used at pilot scale. Decreasing top impeller position improved k(L)a of dCO(2) by 2-fold. Changing headspace aeration rate from 0.02 to 0.04 vvm had no impact on dCO(2) removal. Two pilot runs were conducted using (A) open pipe and (B) antifoam in the presence of sparge stone, both in conjunction with lower impeller position. The presence of antifoam may interfere in product purification; however, demonstration of antifoam removal can be difficult. Open pipe allowed an alternative to using antifoam, as foam level with open pipe was significantly less. Both strategies successfully reduced dCO(2) level by 2.5-fold (179 +/- 9 vs 72 +/- 9 mmHg). Titer at day 10 of culture improved by 1.5-fold. Specific productivity improved by 41%. Historically, cultures were harvested around day 9-11 because of the high amount of foam; both strategies allowed the cultures to be extended up to day 14, resulting in 2-fold higher titer compared to that of the historical control without compromising protein quality.

Strategies for Improved dCO2 Removal in Large-Scale Fed-Batch Cultures

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr™) is an automated micro-bioreactor system with miniature single-use bioreactors with a 10-15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in-line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr™ resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr™ was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr™ system as a high throughput system for cell culture process development.

High-throughput miniaturized bioreactors for cell culture process development: Reproducibility, scalability, and control

https://www.exphem.org/article/S0301-472X(01)00658-0/pdf

Oxygen tension modulates the expression of cytokine receptors, transcription factors, and lineage-specific markers in cultured human megakaryocytes.

http://wolfson.huji.ac.il/purification/Course92632_2014/MixedMode/Wolfe2014.pdf

Sigma S. Mostafa

Papers

Oxygen tension influences the differentiation, maturation and apoptosis of human megakaryocytes

Strategies for Improved dCO2 Removal in Large-Scale Fed-Batch Cultures

High-throughput miniaturized bioreactors for cell culture process development: Reproducibility, scalability, and control

Oxygen tension modulates the expression of cytokine receptors, transcription factors, and lineage-specific markers in cultured human megakaryocytes.

Multimodal chromatography: Characterization of protein binding and selectivity enhancement through mobile phase modulators.