384-Well High-Throughput ELISA Method for Host Cell Protein Analysis

By Sanjukta Chatterjee, Ph.D.
Global Principal Scientist

Developing a 384-Well ELISA Method for HCP Analysis

Host-cell proteins (HCP) are process-related impurities that could present potential safety and quality concerns for biologic drugs. HCP must be controlled during drug manufacturing as this is considered as a Critical Quality Attribute (CQA). The ability to detect HCP during downstream operations is essential to the development of a robust and effective purification process.

Pressure to reduce drug development timelines has been increasing significantly in recent years, while novel molecular formats (NMF) have become more prevalent as drug developers seek to bring more effective medicines to market. Many of these NMFs cannot be purified using platform methods traditionally used for monoclonal antibodies (mAbs). The development of purification processes for these molecules is often challenging, as it is difficult to predict under which conditions the product and impurities will bind or elute. Development of a robust and reliable chromatographic separation requires screening of many different resin chemistries, buffers, and process conditions, generating large numbers of samples that must all be evaluated for host cell protein analysis.

Figure 1: HCP clearance is monitored at each stage of purification

Challenges with Traditional ELISA Methods

To support faster process development, high-throughput screening (HTS) requires HCP analysis of large number of samples. Traditional HCP ELISA methods struggle to efficiently handle large sample numbers (30 to 50) in a timely manner, hindering accelerated process development. As part of the Global Analytical Development team at Lonza, I have had the opportunity to work alongside a skilled team of scientists to develop a practical solution to address this need.

Introducing the 384-Well ELISA Method High-Throughput Approach

Our approach utilizes a 384-well ELISA plate format, enabling the analysis of four times as many samples in a single HCP assay. This advancement not only increases throughput but also significantly reduces the volume requirements for samples and critical reagents. The performance of this method is at par with Lonza’s 96-well plate HCP method, making it a viable and interchangeable alternative. From a regulatory perspective, this consistency supports a robust analytical method lifecycle, enabling smoother method bridging and transfer across sites. It also reinforces confidence in maintaining analytical continuity - a key expectation in regulatory filings and audits.

Figure 2: Comparability of 384-well and 96-well HCP analysis

Overcoming Plate Format Bias and Improving Consistency

One of the most significant challenges in developing a high-throughput ELISA for host cell protein analysis was managing format-related plate effects - where sample recovery varies depending on the well’s position on the plate.

To address this, we collaborated closely with a team of experienced scientists to design a balanced plate layout that neutralizes positional bias. This format ensures consistent HCP recovery across all wells, regardless of sample location, supporting both analytical reliability and regulatory expectations for method robustness and lifecycle consistency.

In parallel, we integrated a benchtop semi-automated liquid handler to streamline pipetting. This eliminated time-consuming and error-prone manual steps, while also reducing critical reagent consumption by 25%. Notably, these gains in throughput and consistency were achieved without the need for complex automation systems, making the method both scalable and accessible for routine use.

Impact on Downstream Processing and Customer Studies

The 384-well ELISA method has been successfully implemented by the Method Development team to support customer studies, and we are pleased to receive encouraging feedback from multiple teams. This high-throughput screening approach significantly shortens the timeline for host cell protein analysis, enabling faster process characterization and downstream process (DSP) development for NMFs. Early access to impurity data during purification development empowers teams to make informed decisions sooner, ultimately saving both time and resources as projects progress.

At Lonza, the Global Analytical Development team plays a key role in identifying challenges across our analytical network and delivering solutions that are impactful on a day-to-day basis. I am proud that we have addressed a long-standing need by developing a cost-effective, high-throughput HCP ELISA method.

I have a decade of experience in the biopharmaceutical sector developing and troubleshooting ELISA methods. This project allowed me to draw on that experience and apply my scientific knowledge to a challenge that has long persisted in process development. It is incredibly rewarding to contribute to a solution that not only enhances our internal capabilities but also strengthens our ability to support customers in delivering life-changing and life-saving therapies to patients in need.

At Lonza we recognize this and have a tailored approach to ensure a seamless analytical method transfer which supports customers’ product success. As discussed here, often one of the biggest challenges from an analytical perspective is host cell protein control and measurement. HCP is an obligatory Critical Quality Attribute (CQA) due to safety and because of this we have an orthogonal approach leveraging both ELISA and orthogonal mass spectrometry technologies to de-risk the transfer as much as possible.