# Chromatography-Free Antibody Purification by Affinity-Phase Separation

> **NIH NIH R44** · ISOLERE BIO, INC. · 2020 · $721,793

## Abstract

Abstract
The objective of this NIH Phase II SBIR proposal is to demonstrate the commercial feasibility of IsoTag™, a
novel cost-effective method to purify monoclonal antibodies (mAbs) that eliminates the need for Protein A (PrA)
chromatography. This objective will be achieved by using a recombinant fusion comprised of a stimulus
responsive elastin-like polypeptide (ELP) that can undergo a reversible soluble to insoluble phase transition,
and the Z-domain (ZD) derived from PrA that binds the constant region of mAbs with high affinity and
specificity. This proposal is motivated by the fact that the first and critical step of mAb purification —PrA
affinity chromatography— has not kept pace with improvements in the recombinant expression level of mAbs,
creating a bottleneck in their production. IsoTag™, which combines affinity capture of mAbs and their isolation
by triggered phase separation with microfiltration, addresses this bottleneck. Upon addition of the ELP-ZD
fusion to clarified cell culture harvest, the ELP-ZD binds the mAb. Upon triggering the phase transition of the
mAb/ELP-ZD with a small amount of kosmotropic salt, the mAb/ELP-ZD complex undergoes liquid-liquid phase
separation into micron-sized droplets, allowing it to be separated from all soluble contaminants by continuous
tangential flow filtration, Next, the pH is lowered to ~4.0, which causes the ZD to lose its affinity for the mAb.
The pure, eluted mAb is in the soluble phase and the ELP-ZD can be regenerated from the insoluble phase. In
the NIH funded Phase I of this project, we have fully optimized the ELP-ZD and the process parameters for
IsoTag™ purification. We have shown that at the bench scale, IsoTag™ can be used to successfully isolate
mAb from mammalian cell culture with yields and contaminant removal that outperform commercially available
alternatives (GE Predictor Plate and Thermo Fisher Nab Spin Column). Having established technical feasibility
and developed a research use only, bench scale product, we plan to validate IsoTag™'s commercial feasibility
for large-scale mAb purification in this Phase II proposal. Our Specific Aims to achieve this objective are: (1)
We will optimize microbial production of the ELP-ZD fusion using fermentation and develop a scalable
purification process. We will then perform a 30 L pilot production run and begin developing an ISO9001 quality
control management protocol. (2) We will validate an assay for quantifying residual ELP-ZD. (3) We will
perform side-by-side purification of the NISTmAb at 30 L scale to compare IsoTag™ to leading PrA resins,
MAbSelect SuRe and Amsphere A3. Last, we will use BioSolve and Matlab software to develop a model for an
interface that takes a customer's inputs and calculates a personalized cost savings by switching to IsoTag™.
The outcome of this project will be compelling data that encourages customer conversion. Isolere's vision is
that IsoTag™ will be a plug-and-play downstream process that allows the indus...

## Key facts

- **NIH application ID:** 9986792
- **Project number:** 5R44GM128557-03
- **Recipient organization:** ISOLERE BIO, INC.
- **Principal Investigator:** Kelli Michelle Luginbuhl
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $721,793
- **Award type:** 5
- **Project period:** 2018-08-01 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9986792

## Citation

> US National Institutes of Health, RePORTER application 9986792, Chromatography-Free Antibody Purification by Affinity-Phase Separation (5R44GM128557-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9986792. Licensed CC0.

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