Introduction

The expression of heterogenous proteins in microbial hosts frequently leads to the formation of insoluble aggregates. Efficient transport of mass and energy, and good fluid mixing are necessary in many process operations. These conditions are often attained by operating in turbulent flow, the onset of which can be promoted by, for example, adding static packing or baffles. In a batch process, external mixing is provided in order to promote heat and mass transfer.

Protein refolding is a key unit operation in many industrial and lab processes that produce recombinant biopharmaceuticals using Escherichia Coli. Yield in this step generally controls overall process yield, and at industrially relevant protein concentrations is limited by aggregation. While most refolding operations are optimized with respect to chemical environment, the physical processes affecting yield have been neglected. In this study, we demonstrate that refolding yield for the model protein lysozyme is dependent on mixing intensity during dilution refolding. This is shown for two different reactor configurations: a standard stirred-tank reactor and a novel oscillatory flow reactor. Protein aggregation represents a major pathway for loss of product during refolding. Aggregation is typically suppressed by refolding at low protein concentrations in a batch system or by utilizing a fed-batch or continuous reactor design, as is appropriate for a kinetic scheme characterized simplistically by competing first-order renaturation and higher-order aggregation. Even after taking precise considerations during reactor scale up, the refolding volumes are often large as protein considerations are typically below 1 mg/ml. Also there are concentration gradients and mixing imperfections present that make the task of scaling up even more difficult. As aggregations intermediates can accumulate with a time constant of seconds following dilution, the concentration gradients within the reactor are expected to affect the overall renaturation.However, numerous studies have been conducted on optimizing the chemical environment while the impact of physical parameters like mixing, is overlooked. There is still evidence that mixing intensity affects the refolding yield, as demonstrated for Lysozyme (Goldberg et al., 1991). Despite these efforts the physical understanding of these efforts is still lacking.

Scale-up Procedure The typical scale up procedures are based on dimensional analysis i.e. we keep a dimensionless number and then increase the reactor size by keeping the selected dimensionless number constant. This number can be any number like Reynolds Number, Power Number, mass transfer parameter etc. depending on the requirement of the process. Since the Scale-up is based on mixing intensity and no concentration gradients, hence the dimensionless number selected is Reynolds Number.

Conventional Design Practices A Stirred Tank Reactor (STR) is a basic mixer which is generally used in the industry. A typical STR assembly consists of vessel, agitator, motor mixer, heating/ cooling inlets and outlets in the

heating/cooling jacket. Direct Dilution refolding in an STR is the common practice in the industry. The disadvantage of using the STR for protein refolding is the protein aggregation that occurs inefficient mixing. To overcome this protein concentrations are kept very low (usually ≤ 0.1 mg/ml) which makes the volume of the reactor quite large leading to more complications in the hydrodynamics of the process. Besides, for effective mixing the flow rate is also kept quite high which leads to shorter residence times and thus leaves concentration gradients in the reactor. It is still being used in the industry only because it is inexpensive and easy to scale up. Thus, the inefficiency in mixing at industrial scales is the primary factor that the trend is shifting towards newer and efficient technologies.

Oscillatory Flow Mixer

With the need of having effective mixing and efficient heat and mass transfer there was a need for newer designs of mixers and reactors. One such type of reactor is the Oscillatory Flow Mixer (OFM). OFMs have evolved over the past decade and are attracting a lot of attention from the bio reactor industry. OFM is a novel continuous mixer in which the tubes fitted with orifice plate baffles have an oscillatory motion superimposed upon the net flow of the process fluid. This combination of baffles and oscillatory motion creates a flow pattern conducive to efficient heat and mass transfer. The flow past these baffles induces vortices which provides both radial and axial mixing. This intensity is varied by changing the amplitude and frequency of mixing of the oscillator that drives the piston. This design leads to better dispersed phase mixing and mass transfer performance. OFMs offer efficient and scalable mixing environments. It finds a wide range of application in chemical and process industries. It can be used in both batch and fed-batch modes and can also be configured to act as a set of CSTRs in series. The intensity of the mixing is as per the need of the user and the residence time can be varied from seconds to hours.

Scale-up Parameters

Oscillatory Reynolds Number describes intensity of mixing applied

Net Reynolds Number describes linear fluid flow

Strouhal Number measures the effective eddy propagation

Where

D is the column diameter

Ρ is the fluid density

Μ is the fluid viscosity

Xo is the oscillation amplitude (m)

F is the oscillation frequency (Hz)

Scale Up

For considering effective mixing the parameter considered for scale up is Reynolds Number. The mixing generally is performed in turbulent region. At Re0 < 400 the flow pattern represents axi-symmetric laminar flow and for Re0 >400 the flow is more turbulent like (Mackley, 1991). The Reynolds number for the scale up was considered to be 1580 for intense mixing. Figure 1 represents the experimental setup used for the lab scale mixing. The H/D ratio of the reactor was also kept constant during scale up.

Parameter Lab Scale Industry Scale (1000 L)Diameter (D) 2.4 cm 48 cmHeight (H) 28 cm 5.6 mOscillation Amplitude 3mm

ωx0 = 3.3 mm rad/secOscillation Frequency (ω) 22 rad/sMaximum Oscillatory Velocity (ωx0) 66 mm rad/sec

Flow velocity .09 ml/min 1.8 ml/min

Table representing the scale up parameters and their values both before and after scale up.

References

1) The Scale-Up of Oscillatory Flow Mixing by Keith B. Smith , Christ's College Cambridge September 1999

2) K. B. Smith, The Scale up of oscillatory mixing, 1999, University of Cambridge.3) Chew T. Lee, A. Mark buswell, anton P. J. Middelberg, The influence of mixing on

lysozyme renaturation during refolding in an oscillatory flow and a stirred-tank reactor, Chemical Engineering Science 57 (2002) 1679 – 1684

4) Shigeo Katoh & Yoshihiro Katoh, Continuous refolding of lysozyme with fed-batch addition of denatured protein solution (2000).