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During early stage development of a therapeutic IgG1 monoclonal antibody, high levels of low molecular weight (LMW) peaks were observed by high performance size-exclusion chromatography and capillary electrophoresis. Further characterization of the LMW peak enriched HPSEC fractions using reversed phase liquid chromatography coupled to mass spectrometry showed these LMW species were 47 kDa and 50 kDa in size. However, the measured masses could not be matched to any fragments resulting from peptide bond hydrolysis. To identify these unknown LMW species, molecular characterization methods were employed, including high-throughput sequencing of RNA. Transcriptomic analysis revealed the LMW species were generated by mis-splicing events in the heavy chain transcript, which produced truncated heavy chain products that assembled with the light chain to mimic the appearance of fragments identified by routine purity assays. In an effort to improve product quality, an optimized purification process was developed. Characterization of the process intermediates confirmed removal of both LMW species by the optimized process. Our study demonstrates that deep-dive analytical characterization of biotherapeutics is critical to ensure product quality and inform process development. Transcriptomic analysis tools can help identify the cause of unknown species, and plays a key role in product and process characterization. [ABSTRACT FROM AUTHOR]

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike protein is of interest for the development of vaccines and therapeutics against COVID-19. Vaccines are designed to raise an immune response against the spike protein. Other therapies attempt to block the interaction of the spike protein and mammalian cells. Therefore, the spike protein itself and specific interacting regions of the spike protein are reagents required by industry to enable the advancement of medicines to combat SARS-CoV-2. Early production methods of the SARS-CoV-2 spike protein receptor binding domain (RBD) were labor intensive with scalability challenges. In this work, we describe a high yielding and scalable production process for the SARS-CoV-2 RBD. Expression was performed in human embryonic kidney (HEK) 293 cells followed by a two-column purification process including immobilized metal affinity chromatography (IMAC) followed by Ceramic Hydroxyapatite (CHT). The improved process showed good scalability, enabling efficient purification of 2.5 g of product from a 200 L scale bioreactor. [ABSTRACT FROM AUTHOR]

The mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the MmpL (mycobacterial membrane protein large) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complex regulatory network, including the TetR family transcriptional regulators Rv3249c and Rv1816. Here we report the crystal structures of these two regulators, revealing dimeric, twodomain molecules with architecture consistent with the TetR family of regulators. Buried extensively within the C-terminal regulatory domains of Rv3249c and Rv1816, we found fortuitous bound ligands, which were identified as palmitic acid (a fatty acid) and isopropyl laurate (a fatty acid ester), respectively. Our results suggest that fatty acids may be the natural ligands of these regulatory proteins. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by these proteins. Binding of palmitic acid renders these regulators incapable of interacting with their respective operator DNAs, which will result in derepression of the corresponding mmpL genes. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters. [ABSTRACT FROM AUTHOR]

Infections caused by bacteria are a leading cause of death worldwide. Although antibiotics remain a key clinical therapy, their effectiveness has been severely compromised by the development of drug resistance in bacterial pathogens. Multidrug efflux transporters--a common and powerful resistance mechanism--are capable of extruding a number of structurally unrelated antimicrobials from the bacterial cell, including antibiotics and toxic heavy metal ions, facilitating their survival in noxious environments. Transporters of the resistance-nodulation-cell division (RND) superfamily typically assemble as tripartite efflux complexes spanning the inner and outer membranes of the cell envelope. In Escherichia coli, the CusCFBA complex, which mediates resistance to copper(I) and silver(I) ions, is the only known RND transporter specific to heavy metals. Here, we describe the current knowledge of individual pump components of the Cus system, a paradigm for efflux machinery, and speculate on how RND pumps assemble to fight diverse antimicrobials. [ABSTRACT FROM AUTHOR]

Background: Spray-drying is considered a promising alternative drying method to lyophilization (freeze-drying) for therapeutic proteins. Particle counts in reconstituted solutions of dried solid dosage forms of biologic drug products are closely monitored to ensure product quality. We found that high levels of particles formed after reconstitution of protein powders that had been spray-dried under suboptimal conditions. Methods: Visible and subvisible particles were evaluated. Soluble proteins in solution before spray-drying and in the reconstituted solution of spray-dried powder were analyzed for their monomer content levels and melting temperatures. Insoluble particles were collected and analyzed by Fourier transform infrared microscopy (FTIR), and further analyzed with hydrogen-deuterium exchange (HDX). Results: Particles observed after reconstitution were shown not to be undissolved excipients. FTIR confirmed their identity as proteinaceous in nature. These particles were therefore considered to be insoluble protein aggregates, and HDX was applied to investigate the mechanism underlying aggregate formation. Heavy-chain complementarity-determining region 1 (CDR-1) in the aggregates showed significant protection by HDX, suggesting CDR-1 was critical for aggregate formation. In contrast, various regions became more conformationally dynamic globally, suggesting the aggregates have lost protein structural integrity and partially unfolded after spray-drying. Discussion: The spray-drying process could have disrupted the higher-order structure of proteins and exposed the hydrophobic residues in CDR-1 of the heavy chain, contributing to the formation of aggregate through hydrophobic interactions upon reconstitution of spray-dried powder. These results can contribute to efforts to design spray-dry resilient protein constructs and improve the robustness of the spray-drying process. [ABSTRACT FROM AUTHOR]

• We introduce a split intein based affinity chromatographic resin and tag system. • Self-cleaving tag and resin system produces recombinant protein of various formats. • Product quality studies demonstrated proper tag cleavage and biological behavior. • Process performance and yield optimization investigated with statistical DoE studies. Affinity tags are frequently engineered into recombinant proteins to facilitate purification. Although this technique is powerful, removal of the tag is desired because the tag can interfere with biological activity and can potentially increase the immunogenicity of therapeutic proteins. Tag removal is complex, as it requires adding expensive protease enzymes. To overcome this limitation, split intein based affinity purification systems have been developed in which a C C -intein tag is engineered into a protein of interest for binding to a N C -intein peptide ligand fixed to a chromatographic support. Tag removal in these systems is achieved by creating an active intein-complex during protein capture, which triggers a precise self-cleavage reaction. In this work, we show applications of a new split intein system, Cytiva™ ProteinSelect™. One advantage of the new system is that the N C -intein ligand can be robustly produced and conjugated to large volumes of resin for production of gram scale proteins. SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager in this work were successfully captured on the affinity resin and scaled 10-fold. Another advantage of this system is the ability to sanitize the resin with sodium hydroxide without loosing the 10–20 g/L binding capacity. Binding studies with IL-1b and IFNAR-1 ECD showed that the resin can be regenerated and sanitized for up to 50 cycles without loosing binding capacity. Additionally, after several cycles of sanitization, binding capacity was retained for the SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager. As with other split intein systems, optimization was needed to achieve ideal expression and recovery. The N-terminal amino acid sequence of the protein of interest required engineering to enable the cleavage reaction. Additionally, ensuring the stability of the C C -intein tag was important to prevent premature cleavage or truncation. Controlling the hold time of the expression product and the prevention of protease activity prior to purification was needed. These results demonstrate the feasibility of the Cytiva™ ProteinSelect™ system to be used in academic and industrial research and development laboratories for the purification of novel proteins expressed in either bacterial or mammalian systems. [ABSTRACT FROM AUTHOR]

Asparagine (Asn) deamidation is a common posttranslational modification in which Asn is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics. We identified a deamidation variant in moxetumomab pasudotox, an immunotoxin Fv fusion protein drug derived from a 38-kDa truncated Pseudomonas exotoxin A (PE38) for the treatment of hairy-cell leukemia. Although the deamidation site, Asn-358, was outside of the binding interface, the modification had a significant impact on the biological activity of moxetumomab pasudotox. Surprisingly, the variant eluted earlier than its unmodified form on anion exchange chromatography, which often leads to the conclusion that it has a higher positive charge. Here we describe the characterization of the deamidation variant with differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry, which revealed that the Asn-358 deamidation caused the conformational changes in the catalytic domain of the PE38 region. These results provide an explanation for why the deamidation affected the biological activity of moxetumomab pasudotox and suggest the approach that can be used for process control to ensure product quality and process consistency. [ABSTRACT FROM AUTHOR]

Strains of the Burkholderia cepacia complex (Bcc) are Gram-negative opportunisitic bacteria that are capable of causing serious diseases, mainly in immunocompromised individuals. Bcc pathogens are intrinsically resistant to multiple antibiotics, including β-lactams, aminoglycosides, fluoroquinolones, and polymyxins. They are major pathogens in patients with cystic fibrosis (CF) and can cause severe necrotizing pneumonia, which is often fatal. Hopanoid biosynthesis is one of the major mechanisms involved in multiple antimicrobial resistance of Bcc pathogens. The hpnN gene of B. multivorans encodes an integral membrane protein of the HpnN family of transporters, which is responsible for shuttling hopanoids to the outer membrane. Here, we report crystal structures of B. multivorans HpnN, revealing a dimeric molecule with an overall butterfly shape. Each subunit of the transporter contains 12 transmembrane helices and two periplasmic loops that suggest a plausible pathway for substrate transport. Further analyses indicate that HpnN is capable of shuttling hopanoid virulence factors from the outer leaflet of the inner membrane to the periplasm. Taken together, our data suggest that the HpnN transporter is critical for multidrug resistance and cell wall remodeling in Burkholderia. [ABSTRACT FROM AUTHOR]

Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually-transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. The MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here report the crystal structure of the inner membrane MtrD multidrug efflux pump, which reveals a novel structural feature that is not found in other RND efflux pumps. [ABSTRACT FROM AUTHOR]

Active efflux of antimicrobial agents is one of the most important strategies used by bacteria to defend against antimicrobial factors present in their environment. Mediating many cases of antibiotic resistance are transmembrane efflux pumps, composed of one or more proteins. The Neisseria gonorrhoeae MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here describe the crystal structure of N. gonorrhoeae MtrE, the outer membrane component of the MtrCDE tripartite multidrug efflux system. This trimeric MtrE channel forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end, indicating that our structure of MtrE depicts an open conformational state of this channel. [ABSTRACT FROM AUTHOR]