Efficient and Selective Bioconjugation Using Surfactants.

The surfactant sodium decanote is used in the drug substance process of Besponsa, an antibody drug conjugate (ADC), to facilitate bioconjugation between activated calicheamicin derivative (linker payload) and inotuzumab (monoclonal antibody). Under the normal conjugation process conditions, sodium decanoate forms micelles and the micelle formation was shown to be critical for the efficient conjugation reaction. Further screening studies indicated that sodium dodecyl sulfate, sodium deoxycholate, and dodecyltrimethylammonium bromide were also able to facilitate the conjugation reaction. While the choice of surfactant and its concentration in the reaction impact the conjugation efficiency, the charge of surfactant and the choice of linker payload influence the conjugated lysine site selectivity. Eight major conjugated lysine sites are observed in Besponsa, as compared to approximately 80 conjugated lysine sites typically observed in conventional lysine-based ADCs.

Characterization of interactions between heparin/glycosaminoglycan and adeno-associated virus.

Adeno-associated virus (AAV) is a key candidate in the development of gene therapy. In this work, we used surface plasmon resonance spectroscopy to study the interaction between AAV and heparin and other glycosaminoglycans (GAGs). Surface plasmon resonance results revealed that heparin binds to AAV with an extremely high affinity. Solution competition studies showed that binding of AAV to heparin is chain length-dependent. AAV prefers to bind full chain heparin. All sulfo groups (especially N-sulfo and 6-O-sulfo groups) on heparin are important for the AAV-heparin interaction. Higher levels of sulfo group substitution in GAGs enhance their binding affinities. Atomic force microscopy was also performed to image AAV-2 in a complex with heparin.

Electron microscopy analysis of a disaccharide analog complex reveals receptor interactions of adeno-associated virus.

Mechanistic studies of macromolecular complexes often feature X-ray structures of complexes with bound ligands. The attachment of adeno-associated virus (AAV) to cell surface glycosaminoglycans (GAGs) is an example that has not proven amenable to crystallography, because the binding of GAG analogs disrupts lattice contacts. The interactions of AAV with GAGs are of interest in mediating the cell specificity of AAV-based gene therapy vectors. Previous electron microscopy led to differing conclusions on the exact binding site and the existence of large ligand-induced conformational changes in the virus. Conformational changes are expected during cell entry, but it has remained unclear whether the electron microscopy provided evidence of their induction by GAG-binding. Taking advantage of automated data collection, careful processing and new methods of structure refinement, the structure of AAV-DJ complexed with sucrose octasulfate is determined by electron microscopy difference map analysis to 4.8Å resolution. At this higher resolution, individual sulfate groups are discernible, providing a stereochemical validation of map interpretation, and highlighting interactions with two surface arginines that have been implicated in genetic studies. Conformational changes induced by the SOS are modest and limited to the loop most directly interacting with the ligand. While the resolution attainable will depend on sample order and other factors, there are an increasing number of macromolecular complexes that can be studied by cryo-electron microscopy at resolutions beyond 5Å, for which the approaches used here could be used to characterize the binding of inhibitors and other small molecule effectors when crystallography is not tractable.

The Race to Develop the Pfizer-BioNTech COVID-19 Vaccine: From the Pharmaceutical Scientists' Perspective.

At the outset of the coronavirus disease 2019 (COVID-19) pandemic, it was clear that a vaccine would be crucial for global health efforts. The Pfizer and BioNTech teams came together in a race against the virus, working to design, test, manufacture, and distribute a safe and efficacious vaccine in record time for people around the world. Here, we provide backstory commentary from the pharmaceutical scientist perspective on the challenges and solutions encountered in the development of the Pfizer-BioNTech mRNA COVID-19 vaccine (BNT162b2; b2; Comirnaty®; tozinameran). We discuss the foundational science that led to the decision to use an mRNA-based approach. We also describe key challenges in the identification of an optimal vaccine candidate and testing in clinical trials, the continuous efforts to improve the vaccine formulation in response to changing global health priorities and facilitate vaccine accessibility, and how vast quantities of vaccine doses were manufactured and safely delivered to every corner of the globe, all without compromising quality, science, and safety. The key to successfully delivering a safe and efficacious vaccine within nine months was a result of extraordinary, real-time, parallel effort and across-the-board collaboration between stakeholders on a global scale.

Characterization of BNT162b2 mRNA to Evaluate Risk of Off-Target Antigen Translation.

mRNA vaccines have been established as a safe and effective modality, thanks in large part to the expedited development and approval of COVID-19 vaccines. In addition to the active, full-length mRNA transcript, mRNA fragment species can be present as a byproduct of the cell-free transcription manufacturing process or due to mRNA hydrolysis. In the current study, mRNA fragment species from BNT162b2 mRNA were isolated and characterized. The translational viability of intact and fragmented mRNA species was further explored using orthogonal expression systems to understand the risk of truncated spike protein or off-target antigen translation. The study demonstrates that mRNA fragments are primarily derived from premature transcriptional termination during manufacturing, and only full-length mRNA transcripts are viable for expression of the SARS-CoV-2 spike protein antigen.

Oligonucleotide mapping via mass spectrometry to enable comprehensive primary structure characterization of an mRNA vaccine against SARS-CoV-2.

Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty, the world's first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with 100% maximum sequence coverage, and a microheterogeneity assessment of 5' terminus capping and 3' terminus poly(A)-tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general.

Development of an icIEF assay for monitoring AAV capsid proteins and application to gene therapy products.

Adeno-associated virus (AAV) gene therapy vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. Methods traditionally used in quality control labs, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), do not provide a complete understanding of capsid viral protein (VP) charge heterogeneity. In the present study, we developed simple, one-step sample preparation and charge-based VP separation using imaged capillary isoelectric focusing (icIEF) for monitoring AAV products. The robustness of the method was confirmed through a design of experiments (DoE) exercise. An orthogonal reverse-phase (RP) HPLC method coupled with mass spectrometry was developed to separate and identify charge species. Additionally, capsid point mutants demonstrate the capability of the method to resolve deamidation at a single site on the viral proteins. Finally, case studies using two different AAV serotype vectors establish the icIEF method as stability indicating and demonstrate that increases in acidic species measured by icIEF correlate with increased deamidation, which, we show, results in decreased transduction efficiency. The addition of a rapid and robust icIEF method to the AAV capsid analytical toolkit enables development and consistent manufacturing of well-characterized gene therapy products.

Subunit dissociation and metal binding by Escherichia coli apo-manganese superoxide dismutase.

Metal binding by apo-manganese superoxide dismutase (apo-MnSOD) is essential for functional maturation of the enzyme. Previous studies have demonstrated that metal binding by apo-MnSOD is conformationally gated, requiring protein reorganization for the metal to bind. We have now solved the X-ray crystal structure of apo-MnSOD at 1.9Å resolution. The organization of active site residues is independent of the presence of the metal cofactor, demonstrating that protein itself templates the unusual metal coordination geometry. Electrophoretic analysis of mixtures of apo- and (Mn2)-MnSOD, dye-conjugated protein, or C-terminal Strep-tag II fusion protein reveals a dynamic subunit exchange process associated with cooperative metal binding by the two subunits of the dimeric protein. In contrast, (S126C) (SS) apo-MnSOD, which contains an inter-subunit covalent disulfide-crosslink, exhibits anti-cooperative metal binding. The protein concentration dependence of metal uptake kinetics implies that protein dissociation is involved in metal binding by the wild type apo-protein, although other processes may also contribute to gating metal uptake. Protein concentration dependent small-zone size exclusion chromatography is consistent with apo-MnSOD dimer dissociation at low protein concentration (K(D)=1×10⁻5 M). Studies on metal uptake by apo-MnSOD in Escherichia coli cells show that the protein exhibits similar behavior in vivo and in vitro.

The structure of the follistatin:activin complex reveals antagonism of both type I and type II receptor binding.

TGF-beta ligands stimulate diverse cellular differentiation and growth responses by signaling through type I and II receptors. Ligand antagonists, such as follistatin, block signaling and are essential regulators of physiological responses. Here we report the structure of activin A, a TGF-beta ligand, bound to the high-affinity antagonist follistatin. Two follistatin molecules encircle activin, neutralizing the ligand by burying one-third of its residues and its receptor binding sites. Previous studies have suggested that type I receptor binding would not be blocked by follistatin, but the crystal structure reveals that the follistatin N-terminal domain has an unexpected fold that mimics a universal type I receptor motif and occupies this receptor binding site. The formation of follistatin:BMP:type I receptor complexes can be explained by the stoichiometric and geometric arrangement of the activin:follistatin complex. The mode of ligand binding by follistatin has important implications for its ability to neutralize homo- and heterodimeric ligands of this growth factor family.

Crystal Structures of PF-06438179/GP1111, an Infliximab Biosimilar.

BACKGROUND: Higher-order structure (HOS) assessment is an important component of biosimilarity evaluations. While established spectroscopic methods are routinely used to characterize structure and evaluate similarity, the addition of X-ray crystallographic analysis to these biophysical methods enables orthogonal elucidation of HOS at higher resolution. METHODS: Crystal structures of the infliximab biosimilar PF-06438179/GP1111 and the reference product Remicade®, sourced from US and European Union markets, were determined and compared to evaluate HOS similarity. Analytical ultracentrifugation studies were conducted to understand reversible self-association. RESULTS: In contrast to more routine spectroscopic methods, the crystal structures enable three-dimensional assessment of complementarity-determining regions and other local regions at near-atomic resolution. The biosimilar structures are highly similar to those of the reference product, as demonstrated visually and though all-atom root-mean-squared deviation measurements. CONCLUSION: The structures provide new insights into the physicochemical properties of the proposed biosimilar and the reference product, further strengthening the 'totality of evidence' in the evaluation of similarity.

Twinned crystals of adeno-associated virus serotype 3b prove suitable for structural studies.

Adeno-associated viruses (AAVs) are leading candidate vectors for gene-therapy applications. The AAV-3b capsid is closely related to the well characterized AAV-2 capsid (87% identity), but sequence and presumably structural differences lead to distinct cell-entry and immune-recognition properties. In an effort to understand these differences and to perhaps harness them, diffraction-quality crystals of purified infectious AAV-3b particles have been grown and several partial diffraction data sets have been recorded. The crystals displayed varying levels of merohedral twinning that in earlier times would have rendered them unsuitable for structure determination, but here is shown to be a tractable complication.

The structures that underlie normal reproductive function.

The mechanisms and physiology of reproductive function have fascinated scientists throughout time. Recent cellular and molecular level structural studies have provided unprecedented insights into reproductive systems and signaling networks. This 'cutting edge' editorial provides a recent example in each of these areas, namely, the anatomical integrity of the follicle, the molecular structure of activin with its binding partners and the molecular regulation of inhibin. These three examples of structure informing function help explain reproductive health and may provide solutions to reproductive disease.

Infliximab crystal structures reveal insights into self-association.

Aggregation and self-association in protein-based biotherapeutics are critical quality attributes that are tightly controlled by the manufacturing process. Aggregates have the potential to elicit immune reactions, including neutralizing anti-drug antibodies, which can diminish the drug's efficacy upon subsequent dosing. The structural basis of reversible self-association, a form of non-covalent aggregation in the native state, is only beginning to emerge for many biologics and is often unique to a given molecule. In the present study, crystal structures of the infliximab (Remicade) Fc and Fab domains were determined. The Fab domain structures are the first to be reported in the absence of the antigen (i.e., tumor necrosis factor), and are consistent with a mostly rigid complementarity-determining region loop structure and rotational flexibility between variable and constant regions. A potential self-association interface is conserved in two distinct crystal forms of the Fab domain, and solution studies further demonstrate that reversible self-association of infliximab is mediated by the Fab domain. The crystal structures and corresponding solution studies help rationalize the propensity for infliximab to self-associate and provide insights for the design of improved control strategies in biotherapeutics development.

Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B).

Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.

Crystal structures of wild-type and mutated cyclophilin B that causes hyperelastosis cutis in the American quarter horse.

BACKGROUND: Hyperelastosis cutis is an inherited autosomal recessive connective tissue disorder. Affected horses are characterized by hyperextensible skin, scarring, and severe lesions along the back. The disorder is caused by a mutation in cyclophilin B. RESULTS: The crystal structures of both wild-type and mutated (Gly6->Arg) horse cyclophilin B are presented. The mutation neither affects the overall fold of the enzyme nor impairs the catalytic site structure. Instead, it locally rearranges the flexible N-terminal end of the polypeptide chain and also makes it more rigid. CONCLUSIONS: Interactions of the mutated cyclophilin B with a set of endoplasmic reticulum-resident proteins must be affected.

Structure of AAV-DJ, a retargeted gene therapy vector: cryo-electron microscopy at 4.5 Å resolution.

AAV-DJ, a leading candidate vector for liver gene therapy, was created through random homologous recombination followed by directed evolution, selecting for in vivo liver tropism and resistance to in vitro immune neutralization. Here, the 4.5 Å resolution cryo-EM structure is determined for the engineered AAV vector, revealing structural features that illuminate its phenotype. The heparan sulfate receptor-binding site is little changed from AAV-2, and heparin-binding affinity is similar. A loop that is antigenic in other serotypes has a unique conformation in AAV-DJ that would conflict with the binding of an AAV-2 neutralizing monoclonal antibody. This is consistent with increased resistance to neutralization by human polyclonal sera, raising the possibility that changed tropism may be a secondary effect of altered immune interactions. The reconstruction exemplifies analysis of fine structural changes and the potential of cryo-EM, in favorable cases, to characterize mutant or ligand-bound complexes.

Structure-function analysis of receptor-binding in adeno-associated virus serotype 6 (AAV-6).

Crystal structures of the AAV-6 capsid at 3Å reveal a subunit fold homologous to other parvoviruses with greatest differences in two external loops. The electrostatic potential suggests that receptor-attachment is mediated by four residues: Arg(576), Lys(493), Lys(459) and Lys(531), defining a positively charged region curving up from the valley between adjacent spikes. It overlaps only partially with the receptor-binding site of AAV-2, and the residues endowing the electrostatic character are not homologous. Mutational substitution of each residue decreases heparin affinity, particularly Lys(531) and Lys(459). Neither is conserved among heparin-binding serotypes, indicating that diverse modes of receptor attachment have been selected in different serotypes. Surface topology and charge are also distinct at the shoulder of the spike, where linear epitopes for AAV-2's neutralizing monoclonal antibody A20 come together. Evolutionarily, selection of changed side-chain charge may have offered a conservative means to evade immune neutralization while preserving other essential functionality.

Crystal structure of the human collagen XV trimerization domain: a potent trimerizing unit common to multiplexin collagens.

Correct folding of the collagen triple helix requires a self-association step which selects and binds α-chains into trimers. Here we report the crystal structure of the trimerization domain of human type XV collagen. The trimerization domain of type XV collagen contains three monomers each composed of four ß-sheets and an α-helix. The hydrophobic core of the trimer is devoid of solvent molecules and is shaped by ß-sheet planes from each monomer. The trimerization domain is extremely stable and forms at picomolar concentrations. It is found that the trimerization domain of type XV collagen is structurally similar to that of type XVIII, despite only 32% sequence identity. High structural conservation indicates that the multiplexin trimerization domain represents a three dimensional fold that allows for sequence variability while retaining structural integrity necessary for tight and efficient trimerization.

The structure of adeno-associated virus serotype 3B (AAV-3B): insights into receptor binding and immune evasion.

Adeno-associated viruses (AAVs) are leading candidate vectors for human gene therapy. AAV serotypes have broad cellular tropism and use a variety of cellular receptors. AAV serotype 3 binds to heparan sulfate proteoglycan prior to cell entry and is serologically distinct from other serotypes. The capsid features that distinguish AAV-3B from other serotypes are poorly understood. The structure of AAV-3B has been determined to 2.6A resolution from twinned crystals of an infectious virus. The most distinctive structural features are located in regions implicated in receptor and antibody binding, providing insights into the cell entry mechanisms and antigenic nature of AAVs. We show that AAV-3B has a lower affinity for heparin than AAV-2, which can be rationalized by the distinct features of the AAV-3B capsid. The structure of AAV-3B provides an additional foundation for the future engineering of improved gene therapy vectors with modified receptor binding or antigenic characteristics.

A pseudo-plaque method for infectious particle assay and clonal isolation of adeno-associated virus.

A colorimetric method has been developed for the detection of adeno-associated virus (AAV) infectious centers in cell culture monolayers. Due to its non-cytopathic nature, AAV has not been amenable to the traditional plaque assay, involving an agar overlay and cellular stains. As a result, an alternate method was required. The pseudo-plaque assay is based on enzyme-catalyzed color development after a fixed cell monolayer is probed with anti-AAV monoclonal antibodies. In spite of chemical fixation, expected to damage the viral genomes and particles, infectious particles can be recovered and amplified for the propagation of viral clones.