Boosting the production of recombinant therapeutic antibodies is crucial in both academic and industry settings. In this work, we investigated the usage of varying signal peptides by antibody V-genes and their roles in recombinant transient production, systematically comparing myeloma and the native signal peptides of both heavy and light chains in 168 antibody permutation variants. We found that amino acids count and types (essential or non-essential) were important factors in a logistic regression equation model for predicting transient co-transfection protein production rates. Deeper analysis revealed that the culture media were often incomplete and that the supplementation of essential amino acids can improve the recombinant protein yield. While these findings are derived from transient HEK293 expression, they also provide insights to the usage of the large repertoire of antibody signal peptides, where by varying the number of specific amino acids in the signal peptides attached to the variable regions, bottlenecks in amino acid availability can be mitigated.

SummaryBoosting the production of recombinant therapeutic antibodies is crucial in both academic and industry settings. In this work, we investigated the usage of varying signal peptides by antibody genes and their roles in recombinant transient production. Comparing myeloma and the native signal peptides of both heavy and light chains in 168 antibody permutation variants, we performed a systematic analysis, finding amino acids counts to be involved in antibody production to construct a model for predicting co-transfection transient recombinant antibody production rates using the HEK293 system. The findings also provide insights into the usage of the large repertoire of antibody signal peptides.

Interest in IgA as an alternative antibody format has increased over the years with much remaining to be investigated in relation to interactions with immune cells. Considering the recent whole antibody investigations showing significant distal effects between the variable (V) and constant (C)- regions that can be mitigated by the hinge regions of both human IgA subtypes A1 and A2, we performed an in-depth mechanistic investigation using a panel of 28 IgA1s and A2s of both Trastuzumab and Pertuzumab models. FcαRI binding were found to be mitigated by the differing glycosylation patterns in IgA1 and 2 with contributions from the CDRs. On their interactions with antigen-Her2 and superantigens PpL, SpG and SpA, PpL was found to sterically hinder Her2 antigen binding with unexpected findings of IgAs binding SpG at the CH2-3 region alongside SpA interacting with IgAs at the CH1. Although the VH3 framework (FWR) is commonly used in CDR grafting, we found the VH1 framework (FWR) to be a possible alternative when grafting IgA1 and 2 owing to its stronger binding to antigen Her2 and weaker interactions to superantigen Protein L and A. These findings lay the foundation to understanding the interactions between IgAs and microbial superantigens, and also guide the engineering of IgAs for future antibody applications and targeting of superantigen-producing microbes.

Immunoglobulin superantigens play an important role in affinity purification of antibodies and the microbiota-immune axis at mucosal areas. Based on current understanding, Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG) and Finegoldia Protein L (PpL) are thought to only bind specific regions of human antibodies, allowing for selective purification of antibody isotypes and chains. Clinically, these superantigens are often classified as toxins and increase the virulence of the producing pathogen through unspecific interactions with immune proteins. To perform an in-depth interaction study of these three superantigens with antibodies, bio-layer interferometry (BLI) measurements of their interactions with a permutation panel of 63 IgG1 variants of Pertuzumab and Trastuzumab CDRs grafted to the six human Vκ and seven human VH region families were tested. Through this holistic and systemic analysis of IgG1 variants with various antibody regions modified, comparisons revealed novel PpL–antibody interactions influenced by other non-canonical antibody known light-chain framework regions, whereas SpA and SpG showed relatively consistent interactions. These findings have implications on PpL-based affinity antibody purification and design that can guide the engineering and understanding of PpL-based microbiota-immune effects.

Micro-fluidized bed has aroused much attention due to its low-cost, intensified-process and fast-screening properties. In this paper, a micro-fluidized bed (15 × 15 mm in cross-section) was designed and fabricated with the use of the stereolithography printing technique, for the investigation of bubbles’ hydrodynamics and comparison of the solids (3D-printed particles VS fungal pellets) fluidization characteristics. In a liquid–gas system, bubble flow regime started from mono-dispersed homogeneous regime, followed by poly-dispersed homogeneous regime, transition bubble regime and heterogeneous bubble regime with increasing gas flowrates from 3.7 mL/min to 32.7 mL/min. The impacts from operating parameters such as gas flowrate, superficial liquid velocity and gas sparger size on bubble size, velocity and volume fraction have been summarized. In liquid–solid fluidization, different solid fluidization regimes for both particles bed and pellets bed were identified. From the bed expansion results, much higher Umf of 7.8 mm/s from pellets fluidization was observed compared that of 2.3 mm/s in particles fluidization, because the hyphal structures of fungal pellets increased surface friction but also tended to agglomerate. The similar R–Z exponent n (5.7 and 5.5 for pellets and particles, respectively) between pellets and particles was explained by the same solid diameter, but much higher Ut of 436 µm/s in particles bed than that of 196 µm/s in pellets bed is a consequence of the higher density of solid particles. This paper gives insights on the development of MFB and its potential in solid processing.