A Practical Guide to Phage- and Robotics-assisted Near-continuous Evolution.

Robotics-accelerated Evolution techniques improve the reliability and speed of evolution using feedback control, improving the outcomes of protein and organism evolution experiments. In this article, we present a guide to setting up the hardware and software necessary to implement Phage- and Robotics-assisted Near-continuous Evolution (PRANCE). PRANCE combines fast phage-based molecular evolution with the ability to run hundreds of independent, feedback-controlled evolution experiments simultaneously. This paper will describe the hardware requirements and setup for PRANCE, including a liquid-handling instrument, a plate reader, auxiliary pumps, heaters, and 3D-printed containers. We describe how to configure the liquid handling robot to be compatible with Python-based open-source software. Finally, we provide suggestions for the first two experiments that can be conducted with a newly constructed PRANCE system that exercises its capabilities and validates that the system is ready to conduct multiplexed evolution. This guide is intended to serve as a handbook for navigating the considerable equipment setup associated with conducting robotics-accelerated evolution.

Column-free optical deconvolution of intrinsic fluorescence for a monoclonal antibody and its product-related impurities.

The quantification of monoclonal antibody (mAb) aggregates and fragments using high pressure liquid chromatography-size exclusion chromatography (HPLC-SEC) typically requires off-line measurements that are time-consuming and therefore not compatible with real-time monitoring. However, it has been crucial to manufacturing and process development, and remains the industrial standard in the assessment of product-related impurities. Here we demonstrate that our previously established intrinsic time-resolved fluorescence (TRF) approach can be used to quantify the bioprocess critical quality attribute (CQA) of antibody product purity at various stages of a typical downstream process, with the potential to be developed for in-line bioprocess monitoring. This was directly benchmarked against industry-standard HPLC-SEC. Strong linear correlations were observed between outputs from TRF spectroscopy and HPLC-SEC, for the monomer and aggregate-fragment content, with R2 coefficients of 0.99 and 0.69, respectively. At total protein concentrations above 1.41 mg/mL, HPLC-SEC UV-Vis chromatograms displayed signs of detector saturation which reduced the accuracy of protein quantification, thus requiring additional sample dilution steps. By contrast, TRF spectroscopy increased in accuracy at these concentrations due to higher signal-to-noise ratios. Our approach opens the potential for reducing the time and labour required for validating aggregate content in mAb bioprocess stages from the several hours required for HPLC-SEC to a few minutes per sample.

Proof-of-concept analytical instrument for label-free optical deconvolution of protein species in a mixture.

The adoption of process analytical technologies by the biopharmaceutical industry can reduce the cost of therapeutic drugs and facilitate investigation of new bioprocesses. Control of critical process parameters to retain critical product quality attributes within strict bounds is important for ensuring a consistently high product quality, but developing the sophisticated analytical technologies required has proven to be a major challenge. Here, we demonstrate a new optical technique for continuous monitoring of protein species as they are eluted from a chromatographic column, even when they fully co-elute with other protein species, without making any assumption about or peak-fitting to the elution profile. To achieve this, we designed and constructed a time-resolved intrinsic fluorescence lifetime chromatograph, and established an analytical framework for deconvolving and quantifying distinct but co-eluting protein species in real time. This proof-of-concept technology has potentially useful applications as a process analytical technology and more generally as an analytical technique for label-free quantification of proteins in mixtures.

Hepatitis B virus sub-genotype A1 infection is characterized by high replication levels and rapid emergence of drug resistance in HIV-positive adults receiving first-line antiretroviral therapy in Malawi.

BACKGROUND: It has been proposed that hepatitis B virus (HBV) sub-genotype A1 infections have mild outcomes and a low risk of drug-resistance among patients infected with human immunodeficiency virus (HIV) receiving lamivudine-containing antiretroviral therapy (ART) without tenofovir in Africa. METHODS: The virologic expression of HBV sub-genotype A1 coinfection was studied over 12 months in HIV-positive adults starting stavudine/lamivudine/nevirapine in Malawi, using Sanger, deep, clonal, and single full-genome sequencing for the sensitive characterization of HBV resistance-associated mutations (RAMs). RESULTS: Among 1117 subjects, 133 (12%) tested HBsAg-positive. After starting ART, retention rates were 96/133 (72%) at 6 months and 54/133 (41%) at 12 months. Based upon the last available follow-up, 92/96 (96%) subjects achieved HIV-1 RNA <40 copies/mL, 48/96 (50%) showed HBV DNA <14 IU/mL, and 24/96 (25%) acquired HBV RAMs. At 6 months, M204I was detected in 8/46 (17%) and 16/17 (94%) subjects using Sanger and deep sequencing, respectively. At 12 months, all viremic patients had multiple resistance and compensatory mutations coexisting on the same HBV genomes. Comparing HBeA-positive (67/133, 50%) with HBeAg-negative subjects, 64/67 (96%) vs 35/66 (55%) showed baseline HBV DNA >2000 IU/mL (P = .0006), 39/47 (17%) vs 9/49 (82%) had persistent HBV DNA detection during follow-up (P < .0001), and 23/47 (49%) vs 2/49 (4%) acquired HBV RAMs (P < .0001). Baseline HBV DNA levels were median 8.1 vs 5.3 log10 IU/mL in subjects with vs those without treatment-emergent RAMs (P < .0001). CONCLUSIONS: HBV sub-genotype A1 infections showed a severe virologic expression in HIV-positive Malawians. The findings strengthen the urgency of interventions to improve ascertainment and management of chronic hepatitis B in the region.

Optically induced thermal gradients for protein characterization in nanolitre-scale samples in microfluidic devices.

Proteins are the most vital biological functional units in every living cell. Measurement of protein stability is central to understanding their structure, function and role in diseases. While proteins are also sought as therapeutic agents, they can cause diseases by misfolding and aggregation in vivo. Here we demonstrate a novel method to measure protein stability and denaturation kinetics, on unprecedented timescales, through optically-induced heating of nanolitre samples in microfluidic capillaries. We obtain protein denaturation kinetics as a function of temperature, and accurate thermodynamic stability data, from a snapshot experiment on a single sample. We also report the first experimental characterization of optical heating in controlled microcapillary flow, verified by computational fluid dynamics modelling. Our results demonstrate that we now have the engineering science in hand to design integrated all-optical microfluidic chips for a diverse range of applications including in-vitro DNA amplification, healthcare diagnostics, and flow chemistry.