Production of Human α-1-Antitrypsin from Transgenic Rice Cell Culture in a Membrane Bioreactor

Abstract

Transgenic plant cell cultures offer a number of advantages over alternative host expression systems, but so far relatively low product concentrations have been achieved. In this study, transgenic rice cells are used in a two-compartment membrane bioreactor (CELLine 350, Integra Biosciences) for the production of recombinant alpha-1-antitrypsin (rAAT). Expression of rAAT is controlled by the rice alpha-amylase (RAmy3D) promoter, which is induced in the absence of sugar. The extracellular product is retained in the bioreactor's relatively small cell compartment, thereby increasing product concentration. Due to the packed nature of the cell aggregates in the cell compartment, a clarified product solution can be withdrawn from the bioreactor. Active rAAT reached levels of 100-247 mg/L (4-10% of the total extracellular protein) in the cell compartment at 5-6 days postinduction, and multiple inductions of the RAmy3D promoter were demonstrated.

Proteolytic andN-Glycan Processing of Humanα1-Antitrypsin Expressed inNicotiana benthamiana

“…Cleavage of the RCL at 354 E has been found to be typical of the lysosomal Cys proteinase cathepsin L (Johnson et al, 1986) and nontarget Ser proteases (Nelson et al, 1998). Considerable amounts of truncated protein were also obtained in previous attempts to express recombinant A1AT in rice (Oryza sativa) cells (Terashima et al, 1999;Huang et al, 2001;Trexler et al, 2002;McDonald et al, 2005), N. benthamiana (Sudarshana et al, 2006), and Nicotiana tabacum chloroplasts (Nadai et al, 2009). Nevertheless, A1AT produced in transgenic tomato (Solanum lycopersicum) plants does not show degradation (Jha et al, 2012).…”

“…Recombinant A1AT has been produced in human and nonhuman cell production systems with limited success (Blanchard et al, 2011;Brinkman et al, 2012;Ross et al, 2012;Lee et al, 2013). The production suffers from two major drawbacks: low expression levels and/or incorrect glycosylation (Garver et al, 1987;Chang et al, 2003;McDonald et al, 2005;Hasannia et al, 2006;Karnaukhova et al, 2006;Plesha et al, 2007;Agarwal et al, 2008;Nadai et al, 2009;Huang et al, 2010;Arjmand et al, 2011;Jha et al, 2012). The mature plasma-derived 52-kD protein has three N-linked glycosylation sites that are mainly decorated with disialylated structures (Kolarich et al, 2006).…”

Improvement of plastic-based disposable bioreactors for plant science needs

“…The limited development of the technology at an industrial scale is due to well-known drawbacks, such as low growth rate, low productivity and/or instability of the productive cell strains and high cost of the traditional bioreactor technology (stainless steel). However, recent publications dealing with the production of recombinant proteins by plant cells indicate a renewed interest in this technology ( Hellwig et al 2004;Girard et al 2004Girard et al , 2006Soderquist and Lee 2005;McDonald et al 2005). Furthermore, Dow AgroSciences received in January 2006 from USDA the world's first regulatory approval for a plant cell-made vaccine.…”

Two new disposable bioreactors for plant cell culture: The wave and undertow bioreactor and the slug bubble bioreactor

“…They have also been used in several recent studies as a possible mean for the production of various recombinant proteins such as antibodies, enzymes, vaccines, and blood factors (Choi et al, 2003;Doran, 2000;Gao et al, 2004;Girard et al, 2004;Hellwig et al, 2004;McDonald et al, 2005;Soderquist andLee, 2005, Sorrentino et al, 2005; Verdelhan des Molles et al, 1999). Plant cell cultures allow fully controlled production and downstream processing, without any risk of foreign gene dissemination in the environment or contamination by mammalian pathogens, and a good public acceptance.…”