Results and Discussion
Surfactant structure influences recovery yield of IgG. In an attempt to reveal any significant relationship between IgG purity/yield and surfactant structure, we studied several different non-ionic surfactant families (Figure 1, B) i.e., Tween, Brij, Triton and Pluronic (triblock-copolymer) detergents. While a hydrophobic anchor in the form of a hydrocarbon chain of varying length is present in Tween, Brij and Triton surfactants it is not present in the same form in Pluronic F-127 triblock copolymer surfactants. Rather the polypropylene central block forms the micellar core, while the polyethylene oxide blocks form the hydrophilic shell (Figure 1, B). The number of ethylene glycol moieties per detergent monomer did not affect the purity of recovered IgG’s (Figure 2, C-D lanes 2-10) but did influence overall yield. The most efficient detergents in each family, from the point of view of overall yield, were found to be: Tween-20 (hIgG- 75%, mouse IgG- 74%); Brij-O20 (hIgG-69%, mouse IgG- 70%); Brij-100 (hIgG-73%, mouse IgG- 70%) and Triton X-100 (hIgG-72%, mouse IgG -69%). Pluronic F-127 was the least efficient (hIgG -42%, mouse IgG -53%) (Figure 2, C-D). This trend was also observed when analyzing pellet composition immediately following the IgG capturing step, though the presence of PEG-6000 in the system, led to diffused migration of the heavy and light chains that made accurate quantification unreliable (Figure 2, A-B lanes 2-10). Here, Tween-20 with the shortest alkyl tail (12 carbons) produced higher overall yields in comparison to other Tween family members with longer alkyl tails (16-18 carbons) (Figure 2, C-D, lanes 2-5). Moreover, results from the Brij family clearly indicated that overall yields increase with increasing number of oxyethylene units. Accordingly, Brij-10 with ~10 oxyethylene units was less efficient than Brij-O20 or Brij-100 containing ~20 or ~100 oxyethylene units, respectively (Figure 2, C-D, lanes 6-8). It seems therefore that process efficiency is determined by several parameters that include both the length of the detergent hydrocarbon tail and the number of oxyethylene groups.
Influence of aggregate size. An additional parameter that may influence total yield is aggregate size. We recently reported that aggregates with size ranging between 100-200 nm do not efficiently capture IgG. 22 It was therefore surprising to find that DLS characterized Pluronic F-127 aggregates as having the largest size (1312 nm) of all the detergents we studied: i.e., Tween (355 - 685 nm), Brij (635 - 683 nm) , Triton X-100 (913 nm) (Figure 3, A-D) . Thus, the larger size of Pluronic F-127 aggregates apparently could not compensate for the block copolymer core/shell micelle structure.
Filtration. In order to be considered for industrial-scale, continuous production flows of therapeutic mAbs12, IgG capturing and extraction from the aggregates would preferably be accomplishedvia filtration rather than centrifugation. We therefore replaced centrifugation steps with filtration at three critical stages in our IgG purification protocol involving Tween-20, Brij-O20 and Triton X-100 surfactants (Figure 4): (i) aggregate incubation with the IgG/BSA mixture in hybridoma serum free media; (ii) removing residual unbound IgG and/or BSA from the aggregates via 20 mM cold NaCl wash; (iii) recovering IgG with extraction buffer: 50 mM Gly in 30 mM NaCl at pH 3.8 during 5 minutes at room temperature. The results clearly indicated that filtration can indeed replace centrifugation. The purity of recovered hIgG (Figure 4, A lanes 2-7) was not different from that obtained with centrifugation (Figure 2, C-D lanes 2, 7, 9) while the overall yields improved and reached values ranging between 80-90% after two consecutive extraction steps (Figure 4, A). Of the three detergents tested, Triton X-100 repeatedly exhibited superiority over Tween-20 and Brij-O20 (Figure 4, A). Thus, a relatively pure Ab preparation can be conveniently and efficiently obtained via filtration using detergent aggregates belonging to three surfactant families. This finding emphasizes the wide scope of working conditions that can be employed with our purification platform.
Since pharmaceutical use of therapeutic mAbs requires these to be monomeric, 31 recovered hIgG obtained with the filtration protocol was analyzed by dynamic light scattering (DLS). The results indicated that purified hIgG is indeed monomeric, as no significant difference in particle size was observed when compared to the control, pure hIgG which had not been subjected to the surfactant aggregate protocol (Figure 4, B). Particle sizes were in agreement with our previous findings32 and ranged between: 10.4-11.0 nm regardless of the detergent used (i.e. Tween-20, Brij-O20, Triton X-100).
Ab specificity. Though IgG purity, overall recovery yields and monomeric state are factors of paramount importance in the development of new Ab purification methodologies, preservation of Ab specificity must also be validated. This question was answered for a specific Ab (an anti-BSA IgG) using the detergent aggregate platform and the filtration protocol. Accordingly, the recovered anti-BSA was subjected to an ELISA assay in order to assess its capability in binding target (BSA). Positive signals in the ELISA assay were observed with all three aggregates (Figure 4, C) and reflected recovery yields similar to those obtained with human or mouse IgG’s using identical detergent aggregates (Figure 4, A). Preservation of IgG specificity is not surprising when considering the presence of at least ≥14 disulfide bonds in all IgG isotypes. 33 The fact that Tween-20, Brij-O20, Brij-100 and Triton X-100 repeatedly demonstrated their utility with polyclonal human and mouse IgG’s suggests that the IgG capturing step is independent of antibody amino acid sequence. This is an important finding since it implies that a single nonionic detergent may provide a purification platform for many different antibodies, regardless of their biological origins. Such a capability is ideal for industrial downstream processing of therapeutic grade mAbs.
Increasing IgG concentration. The fact that mAb production in CHO cells has improved significantly and currently can reach Ab titers greater than 1 mg/mL 34led us to test our filtration protocol at IgG concentrations up to 5 mg/ml (Figure 5). As before, we focused on the three detergents that led to the highest recovery yields via the centrifugation protocol. The results presented in Figure 5 indicate that process efficiency at 3 mg/mL and 5 mg/mL decreases by 5-10% relative to that obtained at 1 mg/mL. Clearly, working at higher IgG concentrations requires a larger number of aggregated micelles. Indeed, good overall yields (~80%), necessitated an increase by a factor of 1.6 and 2.3 (wt/vol) for 3 mg/ml and 5 mg/ml hIgG, respectively. These results are encouraging as they are obtained at current industrial IgG expression levels. 34
Chelator recycling. Recycling of raw material represents an important environmental and economic concern for large-scale pharmaceutical production. We therefore studied the possibility of regenerating the most costly component in our platform, i.e. the chelator (batho). Chelator recycling via crystallization appears to be both economical and realistic due to the inherent physiochemical properties of batho. These include: water-insolubility;35 stability under both acidic 36 and basic conditions, and at elevated temperatures;37 and the ability of batho to crystallize rapidly in aqueous media due to its relatively rigid and extended aromatic system which is capable of formingπ-π interactions and thereby , aromatic stacking between batho molecules.
Tween-20 was the surfactant of choice for this recycling experiment. Initial removal of Tween-20 from the aggregates was essential for optimal batho recycling, as the presence of any detergent would likely improve batho’s water-solubility and hence, suppress its crystallization in aqueous media. However, Tween-20 was found to be soluble even in 1 M NaCl (data not shown). Therefore, Tween-20 aggregates that were used for IgG capture and extraction were thoroughly washed with 0.25 M NaCl to remove the detergent as well as the IgG’s that might not have been extracted from the aggregates. Under these conditions, the extremely hydrophobic [(batho)3:Fe2+] red complex is obviously water-insoluble (Figure 5, Step I). The pellet size decreased after NaCl washing and the resulting, excluded aqueous solution was colorless, indicating no significant loss of the [(batho)3:Fe2+] red complex (data not shown). Light microscopy analysis of washed aggregates showed that these are composed of numerous red crystals (~30-70 µm) (Figure 7, A) containing the [(batho)3:Fe2+] complex as determined by mass spectrometry (MS) (Supplementary S1). In order to dissociate the red complex, crystals were briefly (2-3 min.) heated at 95 °C in DDW/MeOH (1:1 vol/vol) containing 10 mM EDTA. Upon cooling, the red color was no longer observed, being replaced by a colorless, transparent solution (not shown). Thus, the loss of the red color served as a convenient internal indicator for monitoring the rate and efficiency at which the [(batho)3:Fe2+] red complex disintegrates in the presence of the competing EDTA chelator. It should be emphasized that the presence of 50% MeOH in the aqueous medium is essential as it led to the complete dissolution of the red crystals and thus, physical access of EDTA to the chelated iron ions in the [(batho)3:Fe2+] red complex. In order to then completely remove MeOH, which suppresses batho crystallization , the solution was heated to 95 °C for 30 minutes and the resulting precipitate was thoroughly washed with DDW to remove EDTA, the [EDTA-Fe2+] colorless complex and free Fe2+ ions if present (Figure 5, Step IV). Analysis of the precipitate by light microscopy showed that it contains colorless, plate-like crystals (Figure 7, B) while liquid chromatography mass-spectrometry (LC-MS) analysis confirmed that the crystals are indeed >98% pure batho (Supplementary S2, C) identical to , or perhaps even purer than commercial batho crystals prior to use (Supplementary S2, D) (Figure 7, C). The fact that the MS spectrum of recycled batho (Supplementary S2, C) was essentially identical to that of the pure, commercial batho (Supplementary S2, A) implies that, no chemical modifications occurred during chelator regeneration. To further rule out possible chemical modifications of the chelator during the recycling procedure, regenerated batho crystals were dissolved in MeOH and Fe2+ ions were added to generate the [(batho)3:Fe2+] red complex. The characteristic absorbance of the latter (λmax = 530 nm) was compared to the absorption spectrum of a freshly prepared [(batho)3:Fe2+] red complex generated from the commercial (unused) chelator and no marked differences were observed (not shown). This finding provided additional supporting evidence for the preservation of the chelating ability of recycled crystalline batho. The characteristic absorption of the complex at 530 nm served as a means of quantitating chelator regeneration efficiency which was found to be, on average, 95% when tested in10 independent experiments (not shown). It should be emphasized that this value represents the recycling yield relative to the total amount of batho present initially in the system. These findings agree with the physiochemical properties of batho described above and show that near quantitative recycling can be achieved.