Abstract Background Recombinant human NELL- rhNELL- is a potent osteogenic protein with therapeutic potential in regenerative medicine A stable formulation is essential to prevent aggregation during production filling storage and clinical use Methodology A four-stage rational formulation strategy was used identify intrinsic aggregation risks of rhNELL- screen polysorbate- and cyclodextrin-based ...More |Related Solutions: μPulse^®

Abstract
Background
Recombinant human NELL-1 (rhNELL-1) is a potent osteogenic protein with therapeutic potential in regenerative medicine. A stable formulation is essential to prevent aggregation during production, filling, storage, and clinical use.

Methodology
A four-stage rational formulation strategy was used: (1) identify intrinsic aggregation risks of rhNELL-1; (2) screen polysorbate- and cyclodextrin-based formulations to enhance colloidal and conformational stability; (3–4) test lead candidates under agitation, freeze/thaw, pH shifts, and elevated temperature. Analytical techniques included PEG challenge, differential scanning fluorimetry (DSF), isothermal chemical denaturation (ICD), and dynamic light scattering (DLS). Aggregation was assessed via visible particles (VP), opalescence, subvisible particles (SVP, Micro Flow Imaging), SDS-PAGE, and ultra-high performance size exclusion chromatography (UP-SEC).

Results
rhNELL-1 was prone to self-association via hydrophobic and electrostatic interactions. Polysorbate 20 (PS20) and hydroxypropyl beta cyclodextrin (HPB-LB-BCD) improved protein stability. PS20 markedly reduced VP and SVP formation. While HPB-LB-BCD alone did not further reduce SVP beyond PS20, it enhanced thermal stress resistance. PS20 was more effective under agitation.

Conclusions
Two lead formulations containing potassium phosphate/Tris buffer, sorbitol, PS20, and HPB-LB-BCD demonstrated strong resistance to aggregation under multiple stresses. PS20 mitigated interfacial stress, while HPB-LB-BCD suppressed solution-phase aggregation, especially at high temperatures. This systematic approach offers a framework for stabilizing other aggregation-prone proteins. Less |Related Solutions: μPulse^®

Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase composed of factor Xa fXa and fVa The affinity of fXa for fVa is low with assembly and function dependent on phospholipid PL membranes Some snakes have evolved venom versions of fXa that bind to fVa ...More |Related Solutions: Rock Imager^®

Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase, composed of factor Xa (fXa) and fVa. The affinity of fXa for fVa is low, with assembly and function dependent on phospholipid (PL) membranes. Some snakes have evolved venom versions of fXa that bind to fVa with high affinity and efficiently activate prothrombin in the absence of PL. We created a similar high-affinity, PL-independent human prothrombinase with 17 mutations to human fXa (M17). The increase in affinity enabled cryogenic electron microscopy (cryo-EM) structure determination of M17-prothrombinase to a resolution of 3.3 Å. All protein domains were well resolved in the map, except for the Gla domain of fXa. The main contacts involve the serine protease and EGF2 domains of fXa and the A2 and A3 domains of fVa, resulting in the burying of a total surface area of 4,900 Å2. The map is of sufficient quality to resolve side chain interactions, including several key M17 mutations. To aid in the placement of the loop Cterminal to the A2 domain (a2-loop), we solved a high-resolution crystal structure of fXa in complex with a synthetic a2 peptide. The acidic a2-loop interacts with the basic heparin binding site of fXa, involving a conserved antiparallel -strand interaction. The M17-prothrombinase structure is compatible with data from biochemical and mutagenesis research and provides important new insights into the assembly and function of the prothrombinase complex. Less |Related Solutions: Rock Imager^®

Severe acute respiratory syndrome coronavirus SARS-CoV- continues to threaten global health This underpins the need for novel therapeutics against this virus Nonstructural protein Nsp of SARS-CoV- is a multifunctional protein with an essential role in viral replication As such it presents itself as an attractive target for drug discovery Here ...More |Related Solutions: NT8^®

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten global health. This underpins the need for novel therapeutics against this virus. Nonstructural protein 1 (Nsp1) of SARS-CoV-2 is a multifunctional protein with an essential role in viral replication. As such, it presents itself as an attractive target for drug discovery. Here, we describe two crystallographic fragment-screening campaigns against Nsp1, one using the established F2X-Entry Screen and one using a new, chemically and structurally diverse fragment library, which we call the KIT library. Together, 21 hits could be identified from 192 screened fragments, which constitutes the highest hit rate reported for Nsp1 to date. Many hits bind to a key functional region and interact with residues involved in cellular mRNA cleavage, ribosome binding and viral RNA recognition. Furthermore, most of the identified fragments share a common binding mode, providing promising starting points for further optimization into drug-like compounds that can disrupt the role of Nsp1 in viral replication. Less |Related Solutions: NT8^®

Ferritins are a widespread family of proteins involved in iron homeostasis While classic ferritins consist of four -helices and form -meric nanocages related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity Here by analyzing the available genomic data we identify a family of double ...More |Related Solutions: NT8^®

Ferritins are a widespread family of proteins involved in iron homeostasis. While classic ferritins consist of four α-helices and form 24-meric nanocages, related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity. Here, by analyzing the available genomic data, we identify a family of double ferritin-like proteins (DFLPs) composed of two four-helical domains, which arose by duplication of a ferritin fold protein. We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens, TmDFLP and CaDFLP, and show that they form homodimers and bind heme. We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase activity. Furthermore, we show that some DFLPs, including TmDFLP and CaDFLP, are highly likely to be targeted into encapsulin shells. Our work expands the range of known iron metabolism systems and highlights the power of genome mining for discovery of new proteins. Less |Related Solutions: NT8^®

Antibody-drug conjugates ADCs are a promising therapeutic modality that enables the delivery of cytotoxic drugs to the target cells that express the corresponding antigen However the purification of ADCs while ensuring product safety homogeneity and stability is a challenging task due to their complex and fragile structure Size exclusion chromatography ...More |Related Solutions: μPulse^®

Antibody-drug conjugates (ADCs) are a promising therapeutic modality that enables the delivery of cytotoxic drugs to the target cells that express the corresponding antigen. However, the purification of ADCs while ensuring product safety, homogeneity, and stability is a challenging task due to their complex and fragile structure. Size exclusion chromatography (SEC), the conventional method for ADC purification, is time-consuming as it requires multiple column washes and equilibration steps. Moreover, subsequent formulation of ADCs, typically using dead-end filtration (DEF), further complicates the production workflow. We compared SEC+DEF with the µPulse®, a miniaturized and automated tangential flow filtration system, for purification and formulation of ADCs. Quality analysis revealed that both approaches were equally gentle as comparable drug-to-antibody ratios (DARs) and monomer purities were observed in the purified samples. Most importantly, both methods exhibited equivalent cleanup efficiency with a 99.8% reduction in free linker-drug concentration. The endotoxin loads comprised 0.11 EU mg-1 for the µPulse and 0.07 EU mg-1 for SEC+DEF, ensuring validation of the safe application of purified ADCs in living systems. However, the µPulse performed purification and formulation of ADCs simultaneously as compared to SEC+DEF, which required multiple manual interventions. Our results indicate that the µPulse is a gentle, single-step, and walk-away approach for the purification of ADCs. Less |Related Solutions: μPulse^®