Commentary: Multiplex dPCR and SV-AUC are Promising Assays to Robustly Monitor the Critical Quality Attribute of AAV Drug Product Integrity.

Adeno-associated viral (AAV) capsids are an emerging vector technology for a number of novel gene therapy modalities (including transgene delivery and CRISPR gene editing). In this commentary, the proper approach to managing uncertainty (described by Rosenberg et al., 2012) when determining critical quality attributes is stated and applied retrospectively to Peginesatide and prospectively to AAV drug product integrity.

Determination of the SLAMF1 self-association affinity constant with sedimentation velocity ultracentrifugation.

Signaling lymphocytic activating molecule family member 1 (SLAMF1 or CD150) is a cell surface glycoprotein expressed on various immune populations, regulating cell-cell interactions, activation, differentiation, and inflammatory responses and has been suggested as a potential target for inflammatory diseases. Signaling is believed to be mediated by high-affinity homophilic interactions; the recombinant soluble form of SLAMF1 has optimal activity in the range of 20 μg/mL. This contradicts with a rather weak homo-dimerization binding constant (K) value reported previously; however, the analytical approach and data analysis suffered from various technical limitations at the time and therefore warrants re-examination.

Analyzing the weak dimerization of a cellulose binding module by analytical ultracentrifugation.

Cellulose binding modules (CBMs) are found widely in different proteins that act on cellulose. Because they allow a very easy way of binding recombinant proteins to cellulose, they have become widespread in many biotechnological applications involving cellulose. One commonly used variant is the CBM from Clostridium thermocellum.

Characterization of therapeutic antibodies in the presence of human serum proteins by AU-FDS analytical ultracentrifugation.

The preclinical characterization of biopharmaceuticals seeks to determine the stability, state of aggregation, and interaction of the antibody/drug with other macromolecules in serum. Analytical ultracentrifugation is the best experimental method to understand these factors. Sedimentation velocity experiments using the AU-FDS system were performed in order to quantitatively characterize the nonideality of fluorescently labeled therapeutic antibodies in high concentrations of human serum proteins.

GDF11 Treatment Attenuates the Recovery of Skeletal Muscle Function After Injury in Older Rats.

Loss of skeletal muscle mass and function results in loss of mobility for elderly patients. Novel therapies that can protect and/or restore muscle function during aging would have profound effects on the quality of life for this population. Growth differentiation factor 11 (GDF11) has been proposed as a "youthful" circulating factor that can restore cardiac, neural, and skeletal muscle functions in aging animals.

Technical Decision Making With Higher Order Structure Data: Perspectives on Higher Order Structure Characterization From the Biopharmaceutical Industry.

Characterization of the higher order structure (HOS) of protein-based biopharmaceutical products is an important aspect of their development. Opinions vary about how best to apply biophysical methods, in which contexts to use these methods, and how to use the resulting data to make technical decisions as drug candidates are commercialized [Gabrielson JP, Weiss WF IV. J Pharm Sci.

Differential Binding Activity of TGF-β Family Proteins to Select TGF-β Receptors.

Growth differentiation factor-11 (GDF11) and myostatin (MSTN) are highly related transforming growth factor-β (TGF-β) ligands with 89% amino acid sequence homology. They have different biologic activities and diverse tissue distribution patterns. However, the activities of these ligands are indistinguishable in in vitro assays.

Crystal structure of human GDF11.

Members of the TGF-β family of proteins are believed to play critical roles in cellular signaling processes such as those involved in muscle differentiation. The extent to which individual family members have been characterized and linked to biological function varies greatly. The role of myostatin, also known as growth differentiation factor 8 (GDF8), as an inhibitor of muscle differentiation is well understood through genetic linkages.

Development of an ultra-sensitive Simoa assay to enable GDF11 detection: a comparison across bioanalytical platforms.

Background: Four bioanalytical platforms were evaluated to optimize sensitivity and enable detection of recombinant human GDF11 in biological matrices; ELISA, Meso Scale Discovery, Gyrolab xP Workstation and Simoa HD-1. Results & methodology: After completion of custom assay development, the single-molecule ELISA (Simoa) achieved the greatest sensitivity with a lower limit of quantitation of 0.1 ng/ml, an improvement of 100-fold over the next sensitive platform (MSD).

A systematic multitechnique approach for detection and characterization of reversible self-association during formulation development of therapeutic antibodies.

In addition to controlling typical instabilities such as physical and chemical degradations, understanding monoclonal antibodies' (mAbs) solution behavior is a key step in designing and developing process and formulation controls during their development. Reversible self-association (RSA), a unique solution property in which native, reversible oligomeric species are formed as a result of the noncovalent intermolecular interactions has been recognized as a developability risk with the potential to negatively impact manufacturing, storage stability, and delivery of mAbs. Therefore, its identification, characterization, and mitigation are key requirements during formulation development.

Monovalent IgG4 molecules: immunoglobulin Fc mutations that result in a monomeric structure.

Antibodies have become the fastest growing class of biological therapeutics, in part due to their exquisite specificity and ability to modulate protein-protein interactions with a high biological potency. The relatively large size and bivalency of antibodies, however, limits their use as therapeutics in certain circumstances. Antibody fragments, such as single-chain variable fragments and antigen binding-fragments, have emerged as viable alternatives, but without further modifications these monovalent formats have reduced terminal serum half-lives because of their small size and lack of an Fc domain, which is required for FcRn-mediated recycling.

A systematic multitechnique approach for detection and characterization of reversible self-association during formulation development of therapeutic antibodies.

In addition to controlling typical instabilities such as physical and chemical degradations, understanding monoclonal antibodies' (mAbs) solution behavior is a key step in designing and developing process and formulation controls during their development. Reversible self-association (RSA), a unique solution property in which native, reversible oligomeric species are formed as a result of the noncovalent intermolecular interactions has been recognized as a developability risk with the potential to negatively impact manufacturing, storage stability, and delivery of mAbs. Therefore, its identification, characterization, and mitigation are key requirements during formulation development.

SEDVIEW, real-time sedimentation analysis.

The ability to obtain a sedimentation coefficient distribution as the run proceeds, and to get an early idea of the quality of a particular sample, has not been made available in real-time during the run in any of the existing software packages. It is desirable on many occasions to be able to see the number of components present in a sample at an early stage of the run. The ability to ascertain the extent of heterogeneity of sample would help enormously to reduce the amount of time that is necessary to obtain that information.

X-ray scattering study of actin polymerization nuclei assembled by tandem W domains.

The initiation of actin polymerization in cells requires actin filament nucleators. With the exception of formins, known filament nucleators use the Wiskott-Aldrich syndrome protein (WASP) homology 2 (WH2 or W) domain for interaction with actin. A common architecture, found in Spire, Cobl, VopL, and VopF, consists of tandem W domains that tie together three to four actin monomers to form a polymerization nucleus.

X-ray scattering study of activated Arp2/3 complex with bound actin-WCA.

Previous structures of Arp2/3 complex, determined in the absence of a nucleation-promoting factor and actin, reveal its inactive conformation. The study of the activated structure has been hampered by uncontrollable polymerization. We have engineered a stable activated complex consisting of Arp2/3 complex, the WCA activator region of N-WASP, and one actin monomer, and studied its structure in solution by small angle X-ray scattering (SAXS).

Leiomodin is an actin filament nucleator in muscle cells.

Initiation of actin polymerization in cells requires nucleation factors. Here we describe an actin-binding protein, leiomodin, that acted as a strong filament nucleator in muscle cells. Leiomodin shared two actin-binding sites with the filament pointed end-capping protein tropomodulin: a flexible N-terminal region and a leucine-rich repeat domain.

Crystal structure of the actin-binding domain of alpha-actinin-4 Lys255Glu mutant implicated in focal segmental glomerulosclerosis.

Mutations in alpha-actinin-4 have been linked to familial focal segmental glomerulosclerosis (FSGS), a common renal disorder in humans, and produce an apparent increase in the actin-binding affinity of alpha-actinin-4 in vitro. One of the mutations, in particular, Lys255Glu, falls in the middle of the actin-binding interface of the actin-binding domain (ABD). The ABD consists of tandem calponin homology (CH) domains (CH1 and CH2).

Toxofilin from Toxoplasma gondii forms a ternary complex with an antiparallel actin dimer.

Many human pathogens exploit the actin cytoskeleton during infection, including Toxoplasma gondii, an apicomplexan parasite related to Plasmodium, the agent of malaria. One of the most abundantly expressed proteins of T. gondii is toxofilin, a monomeric actin-binding protein (ABP) involved in invasion.

Interactions between the leucine-zipper motif of cGMP-dependent protein kinase and the C-terminal region of the targeting subunit of myosin light chain phosphatase.

Nitric oxide induces vasodilation by elevating the production of cGMP, an activator of cGMP-dependent protein kinase (PKG). PKG subsequently causes smooth muscle relaxation in part via activation of myosin light chain phosphatase (MLCP). To date, the interaction between PKG and the targeting subunit of MLCP (MYPT1) is not fully understood.