Early determination of potential critical quality attributes of therapeutic antibodies in developability studies through surface plasmon resonance-based relative binding activity assessment.

Precise measurement of the binding activity changes of therapeutic antibodies is important to determine the potential critical quality attributes (CQAs) in developability assessment at the early stage of antibody development. Here, we report a surface plasmon resonance (SPR)-based relative binding activity method, which incorporates both binding affinity and binding response and allows us to determine relative binding activity of antibodies with high accuracy and precision. We applied the SPR-based relative binding activity method in multiple forced degradation studies of antibody developability assessment. The current developability assessment strategy provided comprehensive, precise characterization of antibody binding activity in the stability studies, enabling us to perform correlation analysis and establish the structure-function relationship between relative binding activity and quality attributes. The impact of a given quality attribute on binding activity could be confidently determined without isolating antibody variants. We identified several potential CQAs, including Asp isomerization, Asn deamidation, and fragmentation. Some potential CQAs affected binding affinity of antibody and resulted in a reduction of binding activity. Certain potential CQAs impaired antibody binding to antigen and led to a loss of binding activity. A few potential CQAs could influence both binding affinity and binding response and cause a substantial decrease in antibody binding activity. Specifically, we identified low abundance Asn33 deamidation in the light chain complementarity-determining region as a potential CQA, in which all the stressed antibody samples showed Asn33 deamidation abundances ranging from 4.2% to 27.5% and a mild binding affinity change from 1.76 nM to 2.16 nM.

Simultaneous silencing of two different Arabidopsis genes with a novel virus-induced gene silencing vector.

BACKGROUND: Virus-induced gene silencing (VIGS) is a useful tool for functional characterizations of plant genes. However, the penetrance of VIGS varies depending on the genes to be silenced, and has to be evaluated by examining the transcript levels of target genes. RESULTS: In this report, we report the development of a novel VIGS vector that permits a preliminary assessment of the silencing penetrance. This new vector is based on an attenuated variant of Turnip crinkle virus (TCV) known as CPB that can be readily used in Arabidopsis thaliana to interrogate genes of this model plant. A CPB derivative, designated CPB1B, was produced by inserting a 46 nucleotide section of the Arabidopsis PHYTOENE DESATURASE (PDS) gene into CPB, in antisense orientation. CPB1B induced robust PDS silencing, causing easily visible photobleaching in systemically infected Arabidopsis leaves. More importantly, CPB1B can accommodate additional inserts, derived from other Arabidopsis genes, causing the silencing of two or more genes simultaneously. With photobleaching as a visual marker, we adopted the CPB1B vector to validate the involvement of DICER-LIKE 4 (DCL4) in antiviral defense against TCV. We further revealed the involvement of ARGONAUTE 2 (AGO2) in PDS silencing and antiviral defense against TCV in dcl2drb4 double mutant plants. These results demonstrated that DOUBLE-STRANDED RNA-BINDING PROTEIN 4 (DRB4), whose protein product (DRB4) commonly partners with DCL4 in the antiviral silencing pathway, was dispensable for PDS silencing induced by CPB1B derivative in dcl2drb4 double mutant plants. CONCLUSIONS: The CPB1B-based vector developed in this work is a valuable tool with visualizable indicator of the silencing penetrance for interrogating Arabidopsis genes, especially those involved in the RNA silencing pathways.

Shotgun proteomics analysis on maize chloroplast thylakoid membrane.

In this study we initiated a proteomic investigation of the maize thylakoid membrane by using a shotgun proteomic approach based on LC-MS(E). A total of 34 maize thylakoid membrane proteins were identified, the majority of which are primarily involved in photosynthesis, including the light-reaction and carbon assimilation. It is noteworthy that all of the core subunits of the Photosystem II were identified in our search. Proteins involved in other processes, such as iron storage, were also detected in our study. The quantity of each identified protein was also determined. Of interest, we discovered that the amount of the three ATP synthase subunits were not equivalent, suggesting that these proteins perform other functions in addition to ATP synthesis. To our knowledge this is the first extensive proteomic investigation of the maize thylakoid membrane, and will likely enable further study of maize photosynthesis and chloroplast development.

Identification of low abundance polyA-binding proteins in Arabidopsis chloroplast using polyA-affinity column.

Proteins could be well separated and further identified by the use of 2-DE and related techniques. Yet, there are many proteins could not be detected even by more effective dyes because of their inherent low abundance or their low resolution. As a result, polyA-affinity column was used as a method to enrich polyA-binding proteins and then identified by MALDI-TOF-MS. In this study, 23 Arabidopsis chloroplast protein spots coded by 18 genes were identified, and majority of these proteins were classified into three related categories according to their annotations in the Swiss-Prot database, including NAD-, RNA-, and ATP-binding motifs, respectively. The major goal of the present Arabidopsis chloroplast proteomics project was to identify novel polyA-binding proteins or protein isoforms located in Arabidopsis chloroplasts and the specific research of cellular proteins with extremely low transcription levels could be fulfilled.