Full Antigen Kinetics of Fabs Directly from Crude Periplasmic Extracts on an Anti-CH1 Capture Surface

Key Highlights

• Eliminate purification bottlenecks for crude samples such as PPEs

• Leverage high sensitivity to measure poorly expressing clones

• Full kinetics for > 5000 clones in a single week

Introduction

Bacterial expression platforms are a well-established tool in the generation of therapeutic candidates, especially minimal antibody formats such as Fab, ScFv, and VHH^1-5. Key advantages include low cost, rapid growth, and high expression yields that are well suited for early drug discovery where large candidate panels are interrogated for relevant therapeutic profiles. Challenges with bacterial platforms include lack of the breadth of post-translational modifications found in eukaryotic systems as well as difficulties maintaining active and properly folded proteins under certain conditions. While E. Coli remains the de facto bacterial expression platform, alternative systems leveraging Brevibacillus, L. lactis, Pseudomonas, and Streptomyces have been developed, albeit in some cases for more custom expression needs⁶.

As is the case in any therapeutic discovery effort, characterizing the maximum number of candidates at a depth sufficient to drive decision making and meet overall research objectives is key. E. Coli can be leveraged to express large numbers of candidates, but recovery of expressed protein from the periplasm (as periplasmic extracts or PPEs) yields an initial low concentration product that contains impurities relating to outer membrane and periplasmic components. A multitude of techniques are available to recover highly pure protein from PPEs, but these come with additional time and resource inputs for candidates that may ultimately prove to be non-ideal for further progression and scale up.

Aside from the purification requirement for many assays, the sheer number of unique clones that can be generated in an E. Coli expression workflow requires techniques that can handle hundreds to even thousands of candidates. Assays such as ELISA certainly can meet this scale of screening, but they provide simple yes/no outputs and most often fail to deliver on the needed depth of characterization. Conversely many real-time binding techniques can deliver this depth of characterization but are not well adapted to the scale of screening required in early candidate discovery.
To address this, we describe here a workflow for characterizing detailed antigen binding kinetics of Fabs directly from crude PPEs using the Carterra LSA^XT®. To circumvent the need for PPE purification, this approach relies on capture of Fabs on a biosensor surface and subsequent washing away of non-Fab cellular matrices. Following capture, a broad titration of antigen concentrations is serially injected across the sensor surface to develop a comprehensive kinetic profile for each Fab clone. The sensor surface has a capacity of up to 384 clones at a time and can be stripped to allow for a new set of Fabs to be captured, with capacity for full kinetics on up to 1152 Fabs in a single experiment. The general strategy here could be extended as well to other antibody formats, including those from yeast or mammalian sources.

Posted by Noah T. Ditto