In situ product recovery of antibodies with a reverse flow diafiltration membrane bioreactor

Meier, Kristina; Büchs, Jochen (Thesis advisor); Melin, Thomas (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2015)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2015


Continuous processes are established in many industrial sectors such as steel, food or petrochemistry due to their cost effectiveness. Though they are characterized by their high space-time-yields, constant product quality as well as low downtimes, only a few continuous processes were implemented in the biotechnological pharmaceutical industry. This is caused by an increased complexity, a higher contamination risk and extensive regulations. Furthermore, in continuous biochemical engineered processes an additional cell retention system is required to achieve high product yields and to be economical competitive. In this regard, one option is the reverse-flow diafiltration, a membrane-based cell retention system. The membrane module is submerged in the bioreactor broth. Thus, cells are retained in their optimal environment minimizing shear stress and avoiding limitations such as oxygen. Over the submerged membrane product withdrawal and supply of fresh medium is alternated reducing a potential fouling layer. The objective of this work is to establish the reverse-flow diafiltration and modify its settings for different applications.At first, the membrane module was investigated regarding its biocompatibility. As there is no standardized analytical device or method to test biocompatibility, such a test method was developed within this work. The metabolic activity of various microorganisms was determined with the Respiration Activity MOnitoring Systems (RAMOS) as a function of the added amount of polymers commonly applied in biotechnology. Nylon and Polyamide 12, used in cable ties and tubing respectively, were found to delay and inhibit microbial growth. This is caused due to leaching of the plasticizer N-butylbenzenesulfonamide and the monomer 1,8-Diazacyclotetradecane-2,7-dione, respectively, from the polymers. A metabolic activity inhibition threshold concentration between 4 - 10 g L-1 Polyamide 12 tubing and approximately 40 g L-1 Nylon was determined for the cultivation of the yeast Hansenula polymorpha, respectively. After the membrane module was proven to be biocompatible, a configuration of reverse-flow diafiltration was conducted with focus on maximization of space-time-yield and long-term filtration stability. Maximization resulted in an improved 4-step mode of operation. Between each alternation of product solution withdrawal and supply of fresh medium one intermediate step empties the membrane. The two intermediate steps were adjusted and, thus, mixing of permeate and fresh medium could be prevented. Hence, fresh medium was saved while simultaneously dilution of the product solution was avoided. Long-term stability is achieved if the critical flux is not exceeded which is reached at a critical transmembrane pressure increase of 45 Pa min-1. Optimal flux ranges for this process could be identified by a systematic flux step method: The critical flux for the yeast Hansenula polymorpha cultured in minimal Syn6 medium and the mammalian cell line CHO DG44 cultured in Power CHO 2 medium is 21 L m-2 h-1 and 9 L m-2 h-1, respectively. The required membrane area for long-term stable processes is determined based on this data.Reverse-flow diafiltration was successfully applied over a broad range of dilution rates for both, the yeast H. polymorpha secreting a single-chain antibody and the mammalian cell line CHO DG44 secreting a full length antibody. The space-time-yield for H. polymorpha could be tripled in comparison to conventional continuous processes. Antibody transmission was above 80% and viability was constantly above 85% for this experiment. Application of reverse-flow diafiltration for CHO cells yields an antibody transmission between 40 and 60%, which is a typical value for membrane-based cell retention systems in literature. Viability was constantly above 90%. The transmembrane pressure was below the critical value for the culture time of over three weeks in both experiments indicating long-term stability. Thus, reverse-flow diafiltration proved to be an in situ cell retention device appropriate for cultivation of yeast and CHO cells. Conventional reverse-flow diafiltration pulses medium resulting in temporal heterogeneities. In microbial cultures especially the C-source alternates between depletion and excess, which leads to oscillations of the DOT signal. This effect is enforced at increased dilution rates. Artificially induced short-term oxygen limitations for H. polymorpha result in the formation of ethanol and a reduced product concentration of 25%. To overcome this cyclic problem, sequential operation of three membranes is introduced. Thus, quasi-continuous feeding is achieved reducing the oscillation of the DOT signal providing a nearly homogenous culture over time. In this thesis, reverse-flow diafiltration proved to be a fail-safe, long-term stable, easy applicable in situ product recovery process with low investment costs and minimal equipment requirements. Space-time-yields could be enhanced remarkably. RFD proved to be especially suitable for shear sensitive cells or organism prone to oxygen limitation.