The preparation of antibodies for therapy is largely based on the use of platform processes producing monoclonal antibodies in mammalian cell systems. Antibody fragments, such as Fabs (∼57 kDa) comprising two chains each of a variable and constant domain, or Fvs (∼27 kDa) comprising two variable domains as two chains or as an engineered single chain (scFv), retain similar antigen binding capacity as the whole antibody and are suitable for expression in microbial cells such as E. coli; they are now entering late stage clinical testing (Andersen and Reilly, 2004; Holt et al., 2003). Domain antibodies (dAbs, ∼12–15 kDa) are based on a single variable domain from the heavy and light chain. These also exhibit high binding affinity and specificity despite lacking most of the constitutive part of a full antibody (Jespers et al., 2004; Saerens et al., 2012). The opportunity now exists to establish platform processes for the production of various antibody fragments using different cell hosts. Such fragments are likely to behave very differently in a process environment; for example, they tend to be more hydrophobic (Ewert et al., 2003; Nieba et al., 1997) and of reduced mass solubility (Famm et al., 2008; Tanha et al., 2006).
The use of ultra scale-down techniques for the evaluation of process options has been discussed in literature: for centrifugation clarification (e.g., Boychyn et al., 2000, 2001, 2004), for centrifuge dewatering and sediment discharge (Chan et al., 2006; Tustian et al., 2007), for membranes (Ma et al., 2010), for pumps (Zhang et al., 2007), and for filters (Reynolds et al., 2003). Large scale centrifugation performance based on its clarification efficiency can be evaluated at the bench scale through the use of the Σ theory, which allows for comparison between centrifuges of different sizes and, using appropriate calibration factors, different centrifuge types (Ambler, 1959). The ultra scale-down (USD) technique mimicking centrifugation is based on the principle that significant hydrodynamic stress will be encountered by the process material in the centrifuge before it enters the settling region. The level of the shear stress depends on the type of machine used (Boychyn et al., 2004). Shear stress is known to disrupt mammalian cells (Hutchinson et al., 2006; Kamaraju et al., 2010; Tait et al., 2009; Zaman et al., 2009), flocs (Berrill et al., 2008), and precipitates (Bell et al., 1982; Byrne et al., 2002; Hoare et al., 1982). Maybury et al. (2000) observed that there could be a 10–58% error in predicting the capacity of a continuous centrifuge if hydrodynamic stress equivalent to the conditions which prevail in the feed zone was not applied to the process material prior to centrifugation. Rotating disc or capillary devices (Boychyn et al., 2001; Chan et al., 2006; Ma et al., 2002; McCoy et al., 2009; Tait et al., 2009) to mimic the effect of process shear stress may be used. Computational fluid dynamics have been used to help establish correlations between the device and the process shear stress (e.g., Boychyn et al., 2001, 2004), but verification is also achieved experimentally (Hutchinson et al., 2006). The use of flocculating agents to aid the separation of cellular material from fermentation broths is particularly affected by pH, cell concentration, flocculant type (or chemistry), and fluid mechanics (Gasner and Wang, 1970; Wang et al., 1970). E. coli aggregation using cationic polymers occurs through charge redistribution on the surface of originally negatively charged E. coli debris (the charge-mosaic model); here the polymer has to be of sufficient length to ensure positively charged areas are created to induce aggregation (Treweek and Morgan, 1977).
The use of flocculants to enhance the recovery of cells and cell debris has been demonstrated for centrifugation (Milburn et al., 1990; Salt et al., 1995) and for filtration (Aspelund et al., 2008). In addition, a high proportion of nucleic acids, lipids, and colloidal particles are shown to be selectively precipitated from soluble proteins. An ultra scale-down approach was used to optimize the flocculation of E. coli heat lysed cell broth extract using cationic polymer followed by clarification by centrifugation (Berrill et al., 2008) with successful verification at scale using a disc stack centrifuge.
In this paper, the physical characterization of cell broths conditioned by a range of methods including flocculation, are correlated with ultra scale-down methods. This is to help gain a better understanding of their processing.