Integration of continuous precipitation, crystallization and flocculation of recombinant proteins

Increased titer in biopharmaceutical production requires new strategies for economical processing. Precipitation, crystallization and flocculation are unit operation which overcomes productivity limits of chromatography and membrane technology. General engineering principles how to set up a precipitation, crystallization, or flocculation process for purification of recombinant proteins have shown in the past. The biophysical principles of precipitation by salt, organic solvent and non-ionic polymers will be explained and commonality with crystallization and flocculation discussed and it will be shown how they can be integrated into a continuous process for recovery proteins. Thermodynamic (phase diagrams)and engineering models, and kinetics of precipitation, crystallization, and flocculation (orthokinetic and perikinetic phase, induction time) have been developed for several proteins such as antibodies and interferon gamma. Scale up rules will be explained and how a process can be transferred into a continuous operation; in particular, the concept of fractal dimension (Figure 1) and the Camp number discussed. Please click Additional Files below to see the full abstract

Continuous desalting of refolding solution by ion exchange chromatography

Recombinant proteins expressed in E. coli as insoluble inclusion bodies need to be resolubilized and refolded to obtain its native structure. These steps require certain salts, which lead to buffers with elevated conductivity. When loading such a refolding solution on an ion exchange column for capturing only relatively low binding capacities can be achieved. In order to overcome this problem, an additional process step has to be introduced. The traditional approach is dilution, diafiltration or dialysis. Here we present a novel alternative process for salt removal of protein solutions. We applied anion and cation exchangers of a micro-pore type, where only salts can penetrate into the pores, but no proteins, in order to desalt the solution. The columns were connected together to run in a serial setup. In order to increase operation performance, a continuous process was developed comprising of four columns, two anion and two cation exchangers. Continuous mode was achieved by staggered cycling operation, where one set of columns was loaded while the other set was regenerated. Proof of concept using a scFv as model protein was performed. The refolding solution could be successfully deionized resulting in constantly low conductivity below 0.5 mS/cm. By running the process continuously process time could be reduced by 38.5% and at the same time productivity was increased to 163% compared to batch operation. Desalting of the protein solution resulted in 5-7 fold higher binding capacities in subsequent ion exchange capture step by conventional protein binding resins

Modeling the residence time distribution of an end to end integrated biomanufacturing process

With the advancements in continuous manufacturing focused mainly on the development of individual unit operations, only a few end-to-end integrated continuous bioprocesses (ICB) have been reported. As the scope starts shifting also towards commercial applications, detailed process understanding is required for quality process design, process optimization and developing process control strategies. Please click Additional Files below to see the full abstract

Continuous protein precipitation – A robust antibody purification method without the need for steady state conditions during continuous integrated production

Antibody precipitation as capture step is an alternative technology to chromatography, because it is a fully continuous method. Bioreactor effluent from a perfusion reactor can be directly processed without surge tanks and the method can be combined with flocculation. We hypothesized that a continuous precipitation and flocculation reactor can be operated under non-steady state conditions. This will allow the design of reactors with extremely short residence time. In batch precipitation steady stats is achieved when the floc size does not change over time. We developed a continuous capture step for antibodies by protein precipitation with PEG 6000. Precipitation and re-solubilization conditions were established by a high throughput approach in batch mode. Continuous precipitation was realized by a tubular reactor design containing static mixers. The particle size distribution of the product precipitate was on-line monitored by Focused Beam Reflectance Measurement (FBRM) in a flow cell. Various hold times (2 to 20 minutes) of the cell culture harvest and different product concentrations were used to simulate changes during continuous fermentation. Precipitate was collected and separation was performed by centrifugation. High molecular weight impurities were reduced by a factor of 5 to 10, yields of 90% and purity increase by a factor of 2.5 were achieved according to size exclusion chromatography. Using FBRM we were able to demonstrate that different residence times during precipitation do not significantly change the particle size distribution. Stable performance concerning product quality (yield, purity and high molecular weight impurities) for different residence times were demonstrated for up to 90 minutes runtime. The influence of product concentration on the precipitate was quantified by FBRM. We demonstrated that protein precipitation is a robust and feasible capture step for mAb purification that does not require steady state conditions and that such continuous reactors are not compulsory operated at steady state conditions. Optimization potential was identified and can be realized during upscale, and higher yields and better performance can be reasonably expected for larger scales

Residence time distribution of continuous protein a chromatography

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Integration of continuous ethanol precipitation and flocculation into manufacturing of antibodies

Precipitation and flocculation are optimal unit operations for continuous capture of proteins from culture supernatant. Precipitation and flocculation can be operated in a real continuous manner. The methods can be combined and after this capture step the precipitate can be stored as a concentrated solution or even as a precipitate. We have developed several precipitation/ flocculation protocols for capture of antibodies from culture supernatant. These include the combinations of CaCl2 flocculation with either cold ethanol precipitation or PEG precipitation and octanoic acid precipitation with PEG precipitation. The precipitation conditions have been screened in microtiter plates or in case of cold ethanol precipitation in small scale reactors. For cold ethanol precipitation a combination with CaCl2 flocculation is best suited. In the first step the high molecular mass impurities can be removed by flocculation with CaCl2 while in the second step the antibody is precipitated by addition of ethanol and low molecular mass impurities are removed. The whole procedure can be repeated and then final polishing can be performed by an anion exchange step in flow through mode. Octanoic acid precipitation is also a very efficient step but an additional phase can be formed which is difficult to remove. In Figure 1 a bench top reactor for cold ethanol precipitation is depicted. The rector consists of two sections (Figure 1). First a concurrent cooling is installed to ensure a constant cooling rate while ethanol is added to the culture supernatant or pre-treated supernatant. In the second section a countercurrent cooling is installed to keep the reactors at the requested temperature. The industry standard for antibody capture is protein A affinity chromatography. Thus the properties of the antibody after cold ethanol precipitation have been compared to protein A purified material. No significant difference could be observed (Figure 2) in either composition or structure. Further purification has been tested by anion-exchange monoliths in order to remove further host cell proteins. The different protocols will be compared to other standard platforms for antibody purification. The possibilities to integrate in-process control and maintenance of steady state will be discussed. The economics of such a process will be discussed for the different scenarios of clinical phase manufacturing and how strategies can be developed when antibodies become commodities or when oral delivery becomes reality. Next steps will be shown how to scale up such a process

Nanomembranes in biotechnology: Separation of small and large biomolecules

Membranes are widely applied in many industrial areas. However, due to the thickness of the membranes the transport of biomolecules across a membrane is impeded by the relatively long transport times. Nanomembranes, in contrast, provide ultra-fast diffusion times and speed up the transfer of biomolecules significantly. Difficulties in production, the fragility, and hydrophobicity of current nanomembranes prevented their widespread use. However, the key characteristics of nanomembranes are very attractive for a broad variety of applications in e.g. biomedical applications, bioseparation technologies, biosensors, and membrane bioreactors. We have made from hydrophilic polymers in waterproof porous nanomembranes and they are therefore especially suited for use in aqueous media, such as used in biological systems. It can withstand more than tens of thousands time its own weight in water and can be used in a wide pH range and in the presence of a broad range of electrolytes. The porous hydrophilic nanomembranes surface is planar with a thickness between 50-150 nm only with uniform pores and a tensile strength of at least 0.1 MPa. Pore diameter can be adjusted according to application from 10 nm to 500 nm. Currently our porous hydrophilic nanomembranes can be produced on large scale from 1 to 250 cm2. It can additionally comprise (embedded or on the surface) bioactive substances, such as enzymes, substrate receptors, active drugs, etc.. The use of our porous hydrophilic nanomembranes allows for ultra-high speed diffusion of biomolecules, but also selective transport of compounds. Please click Additional Files below to see the full abstract

High cell density culture for VLP production in latent virus-free insect cell line

Continuous virus-like particle (VLP) production in insect cells with the baculo virus expression system faces numerous problems: The presence of defective interfering particles (DIPs) hinders continuous infection, cell growth is slower than virus replication, cell retention membranes and hollow fibres retain VLPs. High cell densities (HCD) can nevertheless enable VLP process intensification in small reactor vessels. We were able to achieve a HCD with the Tnms42 insect cell line in both shaking flasks with pseudo perfusion and in the bioreactor utilizing an alternating tangential flow (ATF) filtration. Compared to some commercially available insect cell lines, this cell line has no persistent adventitious viral infection and is equally well suited to produce HA-GAG VLPs as HighFiveTM cells. With daily medium exchange the exponential growth phase could be elongated and the total cell concentration (TCC) increased by a factor of two in the shaking flask (SF) and four in the bioreactor (BR) to ~40x106 cells/mL in comparison to reference batch processes (~9 Figure 1). Such HCD can be used to optimize and investigate cell concentration at infection (CCI), multiplicity of infection (MOI) and cell state at infection to reach higher volumetric VLP titers. Please click Download on the upper right corner to see the full abstract

Salt tolerant endonucleases for the removal of host cell DNA in Downstream Processing of enveloped viruses

Host cell DNA is a critical impurity in downstream processing of enveloped viruses. For vaccine applications, host cell DNA content should be below 10 ng per dose in the final product. Enveloped viruses exhibit an overall negative net charge on their surface and therefore have binding properties similar to DNA. Consequently, separation of virus from DNA can be cumbersome. In addition to DNA in its naked form, host cell DNA is present in virus preparations in form of chromatin. Chromatin (Figure 1) consists of complex and large structures which include DNA and highly positively charged histones. Therefore, different types of interactions of chromatin with chromatographic material and membranes can be observed, electrostatic interaction through negative charges of DNA and positive charges histones and hydrophobic interaction through hydrophobic patches of histones. Moreover, chromatin is often similar in size to viruses, further complicating their separation. We evaluated the performance of four different endonucleases, two salt tolerant endonucleases and two sensitive to salt, in the downstream processing of recombinant Measles virus. Endonuclease treatment was performed after clarification and followed by a purification step using flowthrough chromatography with Capto™ Core 700 resin. Nanoparticle tracking analysis (NTA) was used to determine size and particle concentration and TCID50 to determine the infectivity of the viruses. DNA and histones presence (in process and purified samples) were determined using PicoGreen™ assay and Western blot analysis using detecting anti-histone antibodies. The salt tolerant endonucleases are more efficient in the removal of chromatin and consequently in the removal of host cell DNA. A 97 % reduction of DNA could be observed. Please click Download on the upper right corner to see the full abstract

Virus-like particles adsorption in anion exchange chromatography media

Biotechnological and pharmaceutical industries have the development of modern vaccines and novel drug delivery systems as one of their main focus. At this point, Virus-Like Particles (VLPs) are key candidates once they have the ability to stimulate humoral and cellular immune responses combined with the inability to replicate or proliferate. VLPs are non-infectious self-assembled protein structures which mimic native viruses (lacking any viral genetic material). However great developments in VLPs manufacturing have already been achieved, their purification is still a complex process, usually slow and with low productivity. Accordingly, there is a demand for new purification strategies and unit operations. Anion exchange chromatography is well established and widely used in industry for the purification all sorts of biomolecules. It is already known that polymer-grafted media in form of charged hydrogels and/or chromatography beads have a very high protein binding capacity and they also bind large biomolecules such as plasmids and viruses. However, the separation mechanism of large biomolecules is still not well understood and this lack of knowledge hinders the development and optimization of the purification processes. To overcome this, our aim is to elucidate the adsorption mechanisms of VLPs, large proteins and protein superstructures into different types of anion exchange chromatography media including highly charged hydrogels and polymer-grafted media. The binding kinetics and equilibria of HIV-1 VLPs expressed in CHO cells and Influenza VLPs expressed in Baculovirus-Insect cell system have been measured for polymer grafted media to elucidate the effect of the charged polymer. Adsorption isotherms were measured in microtiter plates and kinetics in batch mode. Gerster, P., Kopecky, E.-M., Hammerschmidt, N., Klausberger, M., Krammer, F., Grabherr, R., Mersich, C., Urbas, L., Kramberger, P., Paril, T., Schreiner, M., Nöbauer, K., Razzazi-Fazeli, E., Jungbauer, A. Purification of infective baculoviruses by monoliths (2013) Journal of Chromatography A, 1290, 36-45. Jungbauer, A., Hahn, R. Polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies (2008) Journal of Chromatography A, 1184 (1-2), 62-79. Jungbauer, A. Chromatographic media for bioseparation (2005) Journal of Chromatography A, 1065 (1), 3-12