Monitoring of the disulfide scrambled species by mixed-mode SEC-HPLC during the purification of a bispecific antibody.

Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is an analytical method routinely used for assessing aggregation content in protein samples. As SEC-HPLC separates analytes based on their hydrodynamic radius, it generally lacks the capability of differentiating species that are similar in size. Recently while purifying a bispecific antibody (bsAb), we noticed that SEC-HPLC can provide certain degree of resolution between the target bsAb and a disulfide scrambled form, although these two species were identical in molecular weight. In seeing the unexpected potential of SEC-HPLC at resolving species with similar size, we further tested Zenix SEC-300, a mixed-mode SEC-HPLC column from Sepax, which was reported to be capable of separating protein analytes based on other factors besides size. The Zenix column indeed provided resolution much better than the regular SEC-HPLC column. Upon further optimization, the Zenix column allowed close to baseline separation of the correctly folded and the disulfide scrambled species. The current study, as a complement to the previous reports, further demonstrates that mix-mode SEC-HPLC is capable of separating protein analytes that are close in size but are different in conformation and/or surface characteristics.

CaptureSelect FcXP affinity medium exhibits strong aggregate separation capability.

Protein A affinity chromatography has been widely used for initial product capture in recombinant antibody/Fc-fusion purification. However, in general Protein A lacks the capability of separating aggregates (unless the aggregates are too large to enter the pores of resin beads or have their Protein A binding sites buried, in which case the aggregates do not bind). In the current work, we demonstrated that CaptureSelect FcXP affinity medium exhibited strong aggregate separation capability and effectively removed aggregates under pH or conductivity gradient elution in two bispecific antibody (bsAb) cases. For these two cases, aggregate contents were reduced from >16% and >22% (in the feed) to <1% and <5% (in the eluate) for the first and second bsAbs, respectively. While more case studies are required to further demonstrate FcXP's superiority in aggregate removal, findings from the current study suggest that FcXP can potentially be a better alternative than Protein A for product capture in cases where aggregate content is high.

Unravelling the reason for different binding behaviors exhibited by antibody aggregates towards preparative and analytical Protein A columns.

We previously showed that the root cause of low Protein A step yield observed for certain antibodies/Fc-fusions is the presence of non-binding aggregates in cell culture harvest. A pre-assumption for the above conclusion is that the aggregates, while do not bind to the preparative Protein A column, can bind to the analytical Protein A-high performance liquid chromatography (HPLC) column used for titer measurement. In the current work, using materials from a previous case with the low yield issue, we confirmed that non-binding aggregates in preparative Protein A flow-through can indeed bind to the analytical Protein A column. In addition, we showed that this discrepancy is mainly due to the different loading densities applied under these two circumstances. We also demonstrated that aggregate bound to the analytical Protein A column slightly stronger than the monomer, as it exhibited a longer retention time. In summary, the current study not only confirmed that non-binding aggregates detected in the preparative Protein A flow-through bind to the Protein A-HPLC column and contribute to the measured titer of culture harvest but also unravelled the reason for different binding behaviors exhibited by antibody aggregates towards preparative and analytical Protein A columns.

Acidic pH or salt treatment can convert soluble antibody aggregates in culture harvest into monomers and improve Protein A chromatography step yield.

While purifying a regular monospecific antibody, we found that the Protein A step yield was much lower than expected. Further studies revealed that the antibody formed large-size aggregates that did not bind to the Protein A resin, hence leading to dropped recovery. In an attempt to solve this low yield issue, we found that mildly acidic pH or ammonium sulfate treatment can partially convert the aggregates into monomers. In addition, when acidic pH treated culture harvest was processed by Protein A chromatography, the yield was restored to the normal range, suggesting that the monomers recovered from aggregates regained Protein A binding capability. Thus, low pH treatment of culture harvest can be potentially used as a general approach for improving Protein A step yield in cases where non-binding antibody aggregates are formed through noncovalent interactions.

SEC-HPLC analysis of column load and flow-through provides critical understanding of low Protein A step yield.

For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., ∼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this phenomenon. In all cases, existence of non-binding aggregates was confirmed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of Protein A load and flow-through. In addition, we demonstrated that aggregates failed to bind to Protein A resin mainly due to their large sizes, which prevented them from entering the resin beads. As the data suggested, SEC-HPLC analysis of Protein A load and flow-through, although not a standard procedure, can provide information that is critical for understanding the unexpected performance of Protein A chromatography in cases like those being presented here. Thus, SEC-HPLC analysis of Protein A load and flow-through is highly recommended for antibodies/Fc-fusions suffering from low Protein A yield.

Antibody aggregation can lead to reduced Protein A binding capacity and low step yield.

Protein A affinity chromatography has been widely used for antibody capture and initial purification. In general, the yield of this step is relatively high (i.e., >90%). However, recently while purifying a bispecific antibody (bsAb) in appended IgG format, the Protein A capture step experienced low yield (i.e., ∼80%). It was found that the target bsAb started appearing in flow-through at a relatively low load density, suggesting that a portion of the expressed bsAb has compromised Protein A binding capability. Further studies indicated that the bsAb in flow-through was mainly in aggregate form. In addition, normal Protein A step yield was restored when the column was loaded with bsAb monomer. Thus, all the evidence pointed to the fact that aggregate with compromised binding capacity was the cause of low Protein A step yield in this case.

Complementary methods for monitoring hole-hole homodimer associated with a WuXiBody-based asymmetric bispecific antibody.

WuXiBody is a bispecific antibody (bsAb) platform developed by WuXi Biologics. Its key feature is the replacement of one parental antibody's CH1/CL region with the T-cell receptor (TCR) constant domain, which prevents mispairing between non-cognate heavy chain and light chain. In addition, heavy chain heterodimerization in asymmetric WuXiBody molecule is promoted by the knobs-into-holes (KiH) technology. Despite the great success of KiH strategy in improving heterodimer formation, homodimers (especially the hole-hole homodimer) can still be generated at low levels. In general, detection and monitoring of homodimers during KiH bsAb purification are challenging as homodimers share similar physicochemical properties with the target heterodimeric bsAb. Nevertheless, the unique design of WuXiBody allows homodimers to be effectively detected and monitored by multiple methods. In the current work, with an asymmetric WuXiBody case study, we demonstrated that hole-hole homodimer can be effectively monitored by six chromatography methods including hydrophobic interaction chromatography (HIC), reverse phase (RP), cation exchange (CEX), KappaSelect, CaptureSelect CH1-XL and Protein L.

Protein A and CaptureSelect FcXP Affinity Resins Possess the Capability of Differentiating Hole-hole Homodimer Isoforms.

BACKGROUND: Knobs-into-holes (KiH) technology has been widely used in asymmetric bispecific antibody (bsAb) construction to promote heavy chain heterodimerization. However, despite the great improvement of heterodimer formation by this strategy, homodimers (especially the holehole homodimer) can still be generated at low levels. Consequently, hole-hole homodimer is a common byproduct associated with the production of KiH bsAbs. In addition, previous studies showed that hole-hole homodimer exists as two different isoforms. As the major difference between these two isoforms lies in the Fc region, we speculated that Protein A media, which bind IgG Fc region with high affinity, and CaptureSelect FcXP, a CH3 domain-specific affinity resin, may provide certain resolution between these two conformational isoforms. OBJECTIVE: The objective of this study was to study the capability of Protein A and CaptureSelect FcXP affinity resins in differentiating hole-hole homodimer isoforms. METHODS: The hole-hole homodimer was produced in CHO cells by expressing the hole half-antibody. The homodimer, along with the half-antibody was initially captured by Protein A chromatography and was then further purified by size-exclusion chromatography (SEC), which separated the homodimer from the unpaired half-antibody. The purified hole-hole homodimer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analytical hydrophobic interaction chromatography (HIC). The purified hole-hole homodimer was separately processed by columns packed with Protein A and CaptureSelect FcXP resins. The purified hole-hole homodimer was also analyzed by Protein A-high-performance liquid chromatography (HPLC). RESULTS: SDS-PAGE analysis and analytical HIC study confirmed that hole-hole homodimer exists as two conformational isoforms. When the hole-hole homodimer was processed by Protein A and CaptureSelect FcXP chromatography, the elution profiles contained two peaks, indicating that both affinity resins possess the capability of differentiating hole-hole homodimer isoforms. CONCLUSION: Our data suggest that Protein A and CaptureSelect FcXP affinity resins both possess the capability of differentiating hole-hole homodimer isoforms and, therefore, can be used for monitoring isoform conversion under various conditions.

Obtaining acidic and basic charge variants using a twin-column continuous chromatography system.

For recombinantly produced monoclonal antibody (mAb), charge variants including acidic and basic species are common heterogeneities. For characterization purpose, sufficient amount of acidic and basic species with high purity is needed. In this work, we developed an approach that allows for continuous separating and collecting of acidic and basic charge variants. First, with batch-mode cation exchange (CEX) chromatography, the load density and linear salt gradient elution conditions under which good separation of both charge variants can be achieved were determined. Next, a stepwise elution protocol was developed based on the linear gradient elution. Finally, acidic and basic charge variants were persistently produced under stepwise elution using a customized twin-column continuous chromatography system. This approach allows acidic and basic charge variants with high purity (i.e., >90%) to be efficiently generated in sufficient amount, which greatly facilitates the necessary characterization of these mAb variants.

SAC-TRAIL, a novel anticancer fusion protein: expression, purification, and functional characterization.

Recombinant protein pharmaceutical agents have been widely used for cancer treatment. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has broad-spectrum antitumor activity, its clinical applications are limited because most tumor cells eventually develop resistance to TRAIL-induced apoptosis through various pathways. Prostate apoptosis response-4 (Par-4) selectively induces apoptosis in cancer cells after binding to the cell surface receptor, GRP78. In this study, TRAIL was fused with the core domain of Par-4 (SAC) to produce a novel recombinant fusion protein. To obtain solubly expressed fusion protein, a small ubiquitin-related modifier (SUMO) was added to the N-terminus of the target protein. Cytotoxicity assays showed that the purified fusion protein exhibited more significant antitumor activity on cancer cells than that by native TRAIL. The connection order and linker sequence of the fusion proteins were optimized. In vitro cytotoxicity assay showed that the SAC-TRAIL fusion protein, which contained a flexible linker (G4S)3, optimally inhibited the proliferation of cancer cells. Immunofluorescence assays demonstrated that SAC-TRAIL could efficiently and specifically bind to cancer cells. Additionally, circular dichroism assays showed that the secondary structure of the recombinant protein with a flexible linker (G4S)3 has both a lower α-helix and higher random coiling, which facilitates the specific binding of SAC-TRAIL to the receptor. Collectively, these results suggest that the novel recombinant fusion protein SAC-(G4S)3-TRAIL is a potential therapeutic agent for cancer. KEY POINTS: • Improved tumor growth suppression and apoptosis induction potency of SAC-TRAIL. • Enhanced targeting selectivity of SAC-TRAIL in cancer cells. • Lower α-helix and higher random coiling in SAC-TRAIL with flexible linker (G4S)3.

Cadmium sulfide net framework nanoparticles for photo-catalyzed cell redox.

A strategy for synthesizing cadmium sulfide net framework (CdS-NF) nanoparticles was developed in a water-based system under mild reaction conditions. The CdS-NFs have not only the excellent photocatalytic properties of CdS, but also the large surface area and diverse porous structures of a metal-organic framework. An Escherichia coli-CdS-NF hybrid system was constructed using NADH regeneration to promote the conversion of trimethylpyruvate acid to l-tert-leucine. The E. coli-CdS-NF system showed higher NAD+ recycling efficiency and substrate conversion rate than CdS QDs under visible light illumination. This work demonstrates a novel method for developing a brilliant coenzyme recycling photocatalyst in bio-redox reactions.