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Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG-binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin-binding peptide (CBP), and it allows for recovery of 20-30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three-tag system comprised of CBP, streptavidin-binding peptide (SBP) and hexa-histidine. We illustrate the application of this approach for the purification of human Bruton's tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery). Combined with mass spectrometry for protein identification, this TAP strategy facilitated the first nonbiased analysis of Btk interacting proteins. The high efficiency of the SBP-His₆ purification allows for efficient recovery of protein complexes formed with a target protein of interest from a small amount of starting material, enhancing the ability to detect low abundance and transient interactions in eukaryotic cell systems.

Abstract: Antimicrobial peptides are of great interest due to their potential application as novel antibiotics. Large quantities of highly purified peptides are required to meet the needs of basic research and clinical trials. Compared with isolation from natural sources and chemical synthesis, recombinant approach offers the most cost-effective means for large-scale peptide manufacture. Among the systems available for heterologous protein production, Escherichia coli has been the most widely used host. Antimicrobial peptides produced in E. coli are often expressed as fusion proteins, a strategy necessary to mask these peptides’ lethal effect towards the host and protect them from proteolytic degradation. The present article reviews commonly used fusion partners (e.g., solubility-enhancing, aggregation-promoting and self-cleavable carriers, etc.), cleavage methods and optimization options for antimicrobial peptides production in E. coli. In addition, the various approaches developed to generate recombinant human antimicrobial peptide LL-37, which offer excellent examples demonstrating effective production strategies, were briefly discussed. [Copyright &y& Elsevier]

Assembly of IgG-like asymmetric bispecific antibodies (bsAbs) requires heavy chain heterodimerization and cognate heavy-light chain pairings. Multiple strategies have been developed to solve these chain association issues. While these strategies greatly promote correct chain pairing, they normally cannot prevent low amount of chain mispaired byproducts from being generated. Besides, byproducts can also be generated as a result of discordant chain expression. The existence of various byproducts poses considerable challenges to downstream processing during the production of recombinant IgG-like bsAbs. In many cases, yield is greatly compromised for purity improvement. This mini review introduces eight IgG-like bsAb platforms, which share a common feature: they all contain built-in purification-facilitating elements in addition to chain pairing control designs. These platforms, by simultaneously providing solutions to the two issues associated with bsAb production (i.e., correct chain pairing and efficient purification), improve both efficiency and robustness of bsAb production. • Purification of IgG-like bsAb is challenging despite implementation of byproduct-minimizing chain pairing control strategies. • Including purification-facilitating element as a component of the designed bsAb is highly desirable. • Eight bsAb platforms with built-in purification-facilitating elements besides chain pairing control designs are introduced. • These production-efficient bsAb platforms provide guidance and inspiration on design of new platforms. [ABSTRACT FROM AUTHOR]

Abstract: The importance of human LL-37 in host defense and innate immunity is well appreciated as reflected by an exponential increase of relevant literature in Pub-Med. Although several articles reported the expression and purification of this cathelicidin, some protocols suffered from low efficiency in enzyme cleavage of fusion proteins due to aggregation and poor separation of recombinant LL-37 from the carrier protein on reverse-phase HPLC. We present a new method for purifying LL-37 that avoids both problems. In this method, the fusion protein (a tetramer) purified by metal affinity chromatography was readily cleaved at a thrombin site 30-residue upstream of the LL-37 sequence. The released LL-37-containing fragment formed a large soluble aggregate (∼95kDa) at pH ∼7, allowing a rapid and clean separation from the carrier thioredoxin (∼14kDa) by size-exclusion chromatography. Recombinant LL-37 was released from the isolated aggregate by chemical cleavage in 50% formic acid at 50°C for 32h. Due to a dramatic difference in retention time, recombinant LL-37 was well resolved from the S-Tag-containing peptide by RP-HPLC. Compared to previous procedures, the new method involves fewer steps and is highly reproducible. It increases peptide yield by 53%. NMR data support the aggregation of LL-37 into a tetramer with increase of pH as well as the feasibility of structural studies of an isotope-labeled antimicrobial peptide in the lipid micelle of dioctanoyl phosphatidylglycerol (D8PG) for the first time. [Copyright &y& Elsevier]

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. • In general, Protein A affinity chromatography lacks the capability of separating aggregates. • FcXP, a CH3-specific affinity medium, exhibits good aggregate separation potential under linear pH gradient elution. • Linear conductivity gradient, which enables improved resolution, facilitates convenient conversion into stepwise elution. • FcXP can potentially be a better alternative than Protein A for product capture in cases where aggregate content is high. [ABSTRACT FROM AUTHOR]

Transgenic crops express Cry proteins exhibit high resistant to target insect pests. When we evaluate the effects of Cry proteins on the parasitoid of target insect pest via tritrophic experiments (transgenic plant-target insect pest-parasitoid) host quality of parasitoids might decrease because of insecticidal protein ingestion, this would cause host-quality mediated effects and influence the accuracy of biosafety assessment. In the current study, high dose of Cry2Aa protein was injected into the hemolymph of Plodia interpunctella by microinjection, and the hemolymph was used as the carrier to deliver Cry protein to Habrobracon hebetor , which has been previously reported as an ectoparasitoid of P. interpunctella larval, in order to avoid the "host-quality mediated effects". Results showed that injected Cry2Aa remained at high concentration and bioactive in the hemolymph of P. interpunctella parasitized by H. hebetor , the hemolymph of P. interpunctella could be used as carriers of Cry protein to H. hebetor , and high dose of Cry2Aa have no negative impacts on the development time, weight of pupa, sex ratio, adults weight (male and female), adult longevity and fecundity, and the activity of stress-related enzymes of H. hebetor. However, the hemolymph of P. interpunctella injected into Galanthus nivalis agglutinin (the positive control) showed significant negative impact on these parameters measured in the present study of H. hebetor. This indicated that Cry2Aa protein had no detrimental effects on the biological parameters of H. hebetor measured in the current study. Meanwhile, this study provides a new method for the safety evaluation of the ectoparasitoids of target pest and might be expanded to the other species of ectoparasitoids of target insects of Cry proteins in biosafety risk assessment. Image 1 • A simpler and faster biosafety evaluation method for ectoparasitoid larvae on target arthropod of Bt protein. • Cry2Aa has no negative impact on Habrobracon hebetor. • Successfully evaluated the effects of Cry2Aa on ectoparasitoid larvae. [ABSTRACT FROM AUTHOR]

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. • Antibody aggregates that fail to bind preparative Protein A column bind the analytical column used for titer measurement. • The above discrepancy is mainly due to the different loading densities applied under these two circumstances. • Antibody aggregate binds to the Protein A-HPLC column stronger than the monomer, as it exhibits a longer retention time. [ABSTRACT FROM AUTHOR]

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. • Low Protein A step yield has been observed for certain mAbs, bsAbs and Fc-fusions. • SDS-PAGE analysis of Protein A flow-through suggested abnormal binding in these cases but missed important information. • SEC-HPLC analysis of column load and flow-through revealed that poor Protein A performance was due to non-binding aggregates. • Aggregates failed to bind mainly due to their large sizes, which prevented the aggregates from entering the resin beads. • For projects experiencing low Protein A yield, SEC-HPLC analysis of load and flow-through is highly recommended. [ABSTRACT FROM AUTHOR]

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 culture harvest of a monospecific antibody disclosed formation of large- and small-size aggregates. • The large-size aggregates are predominant and did not bind to the Protein A resin, leading to lower-than-expected step yield. • Upon mildly acidic pH or ammonium sulfate treatment, the non-binding aggregates were partially converted into monomers. • Low pH treatment of culture harvest restored normal Protein A yield, suggesting recovered monomers regained binding ability. [ABSTRACT FROM AUTHOR]

Protein A, Protein L and KappaSelect affinity resins have been widely used for antibody purification. Elution of antibody bound to these resins is typically achieved by acidic pH. In addition, elution can be moderately adjusted by tuning the salt concentration in mobile phase as hydrophobic interactions play a major role in binding. In this study, we assessed the impact of salt concentration in mobile phase on antibody retention in these three types of affinity chromatography. The data suggest that salt concentration has a bigger impact on retention in the two light chain-binding affinity columns (i.e., Protein L and KappaSelect) than in Protein A column. In particular, lowering salt concentration in mobile phase for Protein L and KappaSelect columns allows elution become feasible at higher pH. In addition, this finding suggests that wash in these two types of column aimed at removing weakly-bound byproducts can also be performed at increased pH by lowering salt concentration in the wash buffer. Rendering wash and elution feasible at higher pH has practical value for cases where the target antibodies are sensitive to stringent conditions. • Impact of salt concentration on antibody retention in Protein A, Protein L and KappaSelect chromatography is assessed. • The data suggest that salt concentration has a bigger impact on retention in two LC-binding columns than in Protein A column. • By lowering salt concentration, antibody bound to Protein L and KappaSelect can be eluted under milder conditions. • The provided information can potentially benefit antibodies that are sensitive to stringent elution/wash conditions. [ABSTRACT FROM AUTHOR]

For IgG-like bispecific antibodies (bsAbs) whose construction involves sequence engineering to promote desired heavy chain-light chain pairing, ¾ antibody (antibody lacking one light chain) is a frequent byproduct during their recombinant production. As this byproduct shares high similarity in physicochemical properties with the target bsAb, its removal poses a challenge to downstream purification. Capto L is an affinity resin based on Protein L, which binds to the variable region of kappa light chain without interfering with the antigen binding site. In this work, we demonstrated that Capto L provides a convenient means for separating ¾ antibody from the bsAb product. • For bsAb whose construction involves protein engineering to avoid HC-LC mispairing, ¾ antibody is a frequent byproduct. • Removing ¾ antibody can be difficult by commonly used chromatography (e.g., Protein A, IEX, CHT, etc.). • Protein L binds to the variable region of the kappa LC without interfering with the antigen binding site. • Capto L, a Protein L-conjugated affinity resin, provides a straightforward and effective means for ¾ antibody removal. [ABSTRACT FROM AUTHOR]

Asymmetric IgG-like bispecific antibodies (bsAbs) are normally derived from two parental mAbs with different origin. Despite the implementation of heterodimerization-promoting strategy in the design, homodimerization can still occur at a low level during the recombinant production of these molecules. In general, monitoring and removal of homodimers pose great challenges to analytical and purification teams, respectively, as these byproducts share high similarity in physicochemical properties with the target heterodimeric bsAb. Protein L is a bacterial surface protein that binds to the variable region of kappa light chain without interfering with the antigen binding site. In this work, we first showed that different antibodies bind Protein L-conjugated resin with varied strength, and then based on this observation we further demonstrated that Protein L chromatography can be a useful tool for monitoring/separating homodimers during the purification of asymmetric bsAbs. • Homodimers are commonly found byproducts during the recombinant production of asymmetric IgG-like bsAbs. • Monitoring and removal of homodimers pose great challenges to analytical and purification teams, respectively. • Protein L binds to the variable region of the kappa LC without interfering with the antigen binding site. • Protein L chromatography can be a useful tool for monitoring/separating homodimers during the production of asymmetric bsAbs. [ABSTRACT FROM AUTHOR]

Half antibody (one half of an antibody which comprises a heavy chain and a light chain) is a common byproduct in bispecific antibody (bsAb) production and in many cases it represents the major product-related impurity. As half antibody contains only one Fc-domain, it binds Protein A resin weaker than the target bsAb, which contains the full Fc-region. Indeed, Protein A chromatography provides certain resolution between half antibody and the intact bsAb under linear pH gradient elution. Nevertheless, separation between these two species is far from complete under this condition. In this study, we demonstrated that adding salt additive to Protein A mobile phase can significantly improve resolution between half antibody and the intact bsAb, allowing most of the half antibody impurity in the load to be removed by this capture step. Having the majority of half antibody byproduct removed at this early stage is a big advantage as it improves the overall robustness of the downstream process. • A Protein A chromatography process was developed to remove half antibody byproduct associated with bsAb production. • Including 500 mM NaCl in Protein A mobile phase can significantly improve resolution between half antibody and the bsAb. • A salt-containing wash at intermediate pH is the key step for half antibody clearance by Protein A chromatography. • Having the majority of half antibody removed at the capture stage improves the overall robustness of the downstream process. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]