Real-time Bioprocess Monitoring Research Articles

How Not to Make the Joint Extended Kalman Filter Fail with Unstructured Mechanistic Models.

The unstructured mechanistic model (UMM) allows for modeling the macro-scale of a phenomenon without known mechanisms. This is extremely useful in biomanufacturing because using the UMM for the joint estimation of states and parameters with an extended Kalman filter (JEKF) can enable the real-time monitoring of bioprocesses with unknown mechanisms. However, the UMM commonly used in biomanufacturing contains ordinary differential equations (ODEs) with unshared parameters, weak variables, and weak terms. When such a UMM is coupled with an initial state error covariance matrix P(t=0) and a process error covariance matrix Q with uncorrelated elements, along with just one measured state variable, the joint extended Kalman filter (JEKF) fails to estimate the unshared parameters and state simultaneously. This is because the Kalman gain corresponding to the unshared parameter remains constant and equal to zero. In this work, we formally describe this failure case, present the proof of JEKF failure, and propose an approach called SANTO to side-step this failure case. The SANTO approach consists of adding a quantity to the state error covariance between the measured state variable and unshared parameter in the initial P(t = 0) of the matrix Ricatti differential equation to compute the predicted error covariance matrix of the state and prevent the Kalman gain from being zero. Our empirical evaluations using synthetic and real datasets reveal significant improvements: SANTO achieved a reduction in root-mean-square percentage error (RMSPE) of up to approximately 17% compared to the classical JEKF, indicating a substantial enhancement in estimation accuracy.

Raman Spectrometry as a Tool for an Online Control of a Phototrophic Biological Nutrient Removal Process

Real-time bioprocess monitoring is crucial for efficient operation and effective bioprocess control. Aiming to develop an online monitoring strategy for facilitating optimization, fault detection and decision-making during wastewater treatment in a photo-biological nutrient removal (photo-BNR) process, this study investigated the application of Raman spectroscopy for the quantification of total organic content (TOC), volatile fatty acids (VFAs), carbon dioxide (CO2), ammonia (NH3), nitrate (NO3), phosphate (PO4), total phosphorus (total P), polyhydroxyalkanoates (PHAs), total carbohydrates, total and volatile suspended solids (TSSs and VSSs, respectively). Specifically, partial least squares (PLS) regression models were developed to predict these parameters based on Raman spectra, and evaluated based on a full cross-validation. Through the optimization of spectral pre-processing, Raman shift regions and latent variables, 8 out of the 11 parameters that were investigated—namely TOC, VFAs, CO2, NO3, total P, PHAs, TSSs and VSSs—could be predicted with good quality by the respective Raman-based PLS calibration models, as shown by the high coefficient of determination (R2 > 90.0%) and residual prediction deviation (RPD > 5.0), and relatively low root mean square error of cross-validation. This study showed for the first time the high potential of Raman spectroscopy for the online monitoring of TOC, VFAs, CO2, NO3, total P, PHAs, TSSs and VSSs in a photo-BNR reactor.

Chemical and Biological Sensor Capsules for Real-Time Measurement of Cell Properties in Bioreactors

Introduction Real-time bioprocess monitoring and control is needed for the scalable production and deployment of cancer cell therapies at reasonable cost. For example, CAR-T cell therapy shows promise as an effective treatment for cancer with high (83%) remission rates.1 However, the cost of these treatments is too expensive for their mass adoption.2 One reason for the steep price is the difficulty scaling production while ensuring delivery of an efficacious therapy to the patient. One factor preventing scalability is the lack of effective process analytical technology. As a potential solution to this issue, we have developed a fully integrated, wireless, 3D-printed sensor capsule to be used for multiplexed sensing of critical quality attributes (CQAs), namely pH, glucose, and lactate concentrations within cell bioreactors. CQAs are used not only as metrics for cell viability, but also as determinants of a cell’s ability to deliver efficacious treatment. Unlike current process monitoring technology, capsule sensors are buoyant and can be propelled using impellers within the bioreactor. This allows for measurements uniformly inside the bioreactor. To our knowledge, this is the first time that 3D-printed, wireless sensor capsules have been developed for use in cell bioreactors for cancer cell therapy. Methods The capsule consists of electrochemical sensors, and read-out and wireless transmission electronics integrated into a capsule made of a biocompatible polymer (Figure 1). The different sensors for glucose, lactate, and pH along with an Ag on-chip reference electrodes were fabricated using standard lithographic techniques on silicon substrates. The reference electrode consisted of a thin film of Ag deposited by e-beam evaporation that was later coated with polyvinyl butyral (PVB) to improve stability.3 Glucose and lactate sensing was achieved amperometrically using enzymes, glucose oxidase or lactase oxidase respectively, immobilized on a Pt working electrode biased to 0.7 V. pH sensing was achieved by depositing a pH-sensitive oxide on a gold electrode. A pH-sensitive Al2O3 or HfO2 layer was deposited through atomic layer deposition.Figure 1 shows the capsule and sensor design. The capsule packaging was 3D printed using a Stratasys Connex 350 and was designed to be fully insulated from solution except for an opening on the top revealing the various sensors. The sensor chip is installed within the cap of the capsule which can be screwed off, but with wired connection to the electronics housed in the body of the capsule. This allows for simple replacement of the multiplexed sensor chip while retaining the capsule electronics, rechargeable battery, and packaging for reuse. Results and Conclusions Before testing within the capsule, the sensors were tested using microfluidics. Figure 2 shows the performance of the Ag/PVB on-chip pseudo reference electrode (pseudo-RE). The pseudo-RE shows a stable potential in different pH buffer solutions and shows similar sensitivity results for pH sensing when compared to a commercial reference electrode. By having a stable microfabricated pseudoRE, the sensing components will not be a limiting factor for miniaturization of the capsule. Figure 3 shows the response of the pH, lactate, and glucose sensors at different concentrations. The pH sensor shows high sensitivity (~53 mV/pH) over the range of pH 3 to 7. Glucose and lactate sensing was tested in 1X PBS. The glucose sensor shows a linear response to glucose concentrations from 0 to 25 mM validating the use of the sensor within cell culture media.4 The glucose sensor shows the same linear range when tested in human mesenchymal stem cell culture. In this case, the glucose concentration was first tested using a commercial sensor and then diluted to targeted glucose concentrations by dilution with PBS. The lactate sensor shows sensitivity in the concentration range of 1 to 5 mM with saturation occurring around ~6 mM. The capsule’s wireless sensing capability was validated using pH sensing in a beaker. The pH sensing results in Figure 4 were obtained wirelessly from the capsule and were nearly identical to the results measured using our microfluidic system and conventional parameter analyzer.This work has successfully demonstrated wireless sensing using a 3D-printed sensor capsule and functionality of electrochemical sensors for multiple CQAs for cell growth in bioreactors. These sensor capsules can enable scalability of the cell manufacturing process while ensuring delivery of an efficacious treatment. Future work will focus on capsule sensor validation in cell growth media and eventually monitoring of cell growth in bioreactors.

Biography of PharmaceuticalBioprocesses

The use of spectroscopic sensors for bioprocess monitoring is a powerful tool within the process analytical technology (PAT) initiative of the US Food and Drug Administration. Spectroscopic sensors enable the simultaneous real-time bioprocess monitoring of various critical process parameters including biological, chemical, and physical variables during the entire biotechnological production process. Approximately 30 ago, when modern biotechnology provided biological like Nobelmedaled monoclonal antibodies and more for the first time this innovative technology boosted pharm a’s pipeline of drug candidates. However, the low-hanging fruits were early gathered and another disruptive innovation is not in sight. What is often described as the so-called innovation gap gets even worse when looking at the remaining patent terms of the drugs on the market. It becomes shorter and shorter and it is foreseeable that the pipeline of new NCE patents will not balance the loss. On the other hand, this development pushes the market for generics, which in the USA, is believed to exceed $81.5 billion with an impressive projected annual growth rate of approximately 10% for 2011–2013. Consequently, the valuation of big pharm companies is falling in an alarming way. According to Burrell& Company, the 17 most relevant players are valued at only $1 trillion today, compared with $1.6 trillion in 2000. Single-use bioreactors are commonly used in the biopharmaceutical industry today; however, they are mostly limited to mammalian cell culture processes. For microbial processes, concepts including the CELL-tainer® technology provide comparable oxygen mass transfer such as in stirred tank reactors. this type of single-use bioreactor is applicable in biopharmaceutical processes, and also in a seed train for bulk chemicals production such as amino acid production. It is expected that single-use technologies will be applied ever more frequently in microbial-fed batch cultivation processes in combination with improved monitoring and control. Bioprocess and biocatalyst IP developed for small-volume/high value pharmaceutical production will therefore find another even bigger market in bulk volume/ lower value processes. Therefore, today’s financing climate for R&D targeting production processes is very different to the early days of biopharmaceuticals. For example, the development of genome sequencing has been financed at first by pharmaceutical industry investors and related industries. Later, such technologies were used by the chemical industry in their desire for modern biotechnological processes, without ever contributing to the basic cost of development. The controversy over intellectual property rights for pharmaceuticals and access to antiretroviral therapies in developing countries has been the subject of much public debate recently. This article provides a broader context for the debate. It first reviews characteristics of the developing country market for pharmaceuticals, including small markets, distinct disease environments and weak health care and regulatory systems. Pharmaceutical use is sometimes suboptimal due to pricing above marginal cost and positive treatment externalities for infectious diseases; sometimes too great due to the failure of consumers to take into account externalities from drug resistance; and sometimes simply inappropriate due to information asymmetries between health care providers and their patients. Drug procurement is often inefficient and corrupt, and inappropriate regulation can hinder access. In addition, health care workers are politically powerful relative to patients. Developed countries and international organizations could encourage differential pricing, allow more favourable tax treatment of appropriate drug donations, and encourage R&D and Pharmaceuticals and the Developing World 87 facilitate access to new products by committing in advance to purchase products needed in developing countries if and when they are developed.

Real-time bioprocess monitoring using a mid-infrared fibre-optic sensor

This work proposes a simple, easy-to-fabricate and cost-efficient sensor based on mid-infrared (MIR) spectroscopy for in-situ real-time bioprocess monitoring. A process involving the production of sophorolipid biosurfactants by way of fermentation was studied with the aim of monitoring varying substrate and product concentrations over time and also distinguishing between the main two structurally different types of biosurfactant produced: lactonic and acidic sophorolipids. First, measurements were carried out using ATR-FTIR spectroscopy. This was followed by comparative measurements using a fabricated U-bent fibre-optic sensor designed for in-situ monitoring. It was demonstrated for the first time that varying concentrations of sophorolipids in a fermentation broth could successfully be monitored over time using an in-situ MIR fibre-optic sensor. Moreover, acidic and lactonic sophorolipids could simultaneously be identified at varying concentrations. The utilisation of such a sensor in bioprocesses can provide a wealth of real-time data, allowing for improved control of feeding rates and better process characterisation, providing significant opportunities for bioprocess control, resulting in increased production and decreased overall costs.

Technology outlook for real-time quality attribute and process parameter monitoring in biopharmaceutical development-A review.

Real-time monitoring of bioprocesses by the integration of analytics at critical unit operations is one of the paramount necessities for quality by design manufacturing and real-time release (RTR) of biopharmaceuticals. A well-defined process analytical technology (PAT) roadmap enables the monitoring of critical process parameters and quality attributes at appropriate unit operations to develop an analytical paradigm that is capable of providing real-time data. We believe a comprehensive PAT roadmap should entail not only integration of analytical tools into the bioprocess but also should address automated-data piping, analysis, aggregation, visualization, and smart utility of data for advanced-data analytics such as machine and deep learning for holistic process understanding. In this review, we discuss a broad spectrum of PAT technologies spanning from vibrational spectroscopy, multivariate data analysis, multiattribute chromatography, mass spectrometry, sensors, and automated-sampling technologies. We also provide insights, based on our experience in clinical and commercial manufacturing, into data automation, data visualization, and smart utility of data for advanced-analytics in PAT. This review is catered for a broad audience, including those new to the field to those well versed in applying these technologies. The article is also intended to give some insight into the strategies we have undertaken to implement PAT tools in biologics process development with the vision of realizing RTR testing in biomanufacturing and to meet regulatory expectations.

Chemical and Biological Sensor Capsules for Real-Time Measurement of Cell Properties in Bioreactors

Introduction Real-time bioprocess monitoring and control is needed for the scalable production and deployment of cancer cell therapies at reasonable cost. For example, CAR-T cell therapy shows promise as an effective treatment for cancer with high (70-90%) remission rates.1 However, the cost of these treatments is too expensive for their mass adoption.2 One reason for the steep price is the difficulty scaling production while ensuring delivery of an efficacious therapy to the patient. One factor preventing scalability is the lack of effective process analytical technologies. As a potential solution to this issue, we have developed a fully integrated, wireless, 3D-printed sensor capsule to be used for multiplexed sensing of critical quality attributes (CQAs), namely pH, glucose, lactate, and IL-2 concentrations within cell bioreactors. CQAs are used not only as metrics for cell viability, but also as determinants of a cell’s ability to deliver efficacious treatment. Unlike current process monitoring technology, capsule sensors are buoyant and can be propelled using impellers within the bioreactor. This allows for measurements uniformly inside the bioreactor. To our knowledge, this is the first time that 3D-printed, wireless sensor capsules have been developed for use in cell bioreactors for cancer cell therapy. Methods The capsule consists of electrochemical sensors as well as data acquisition and wireless transmission electronics integrated into a capsule made of a biocompatible polymer (Figure 1). The sensors for glucose, lactate, pH, and IL-2 along with Ag on-chip pseudo-reference electrode (pseudo-RE) were fabricated using standard lithographic techniques on silicon substrates. The reference electrode consisted of a thin-film of Ag that was later coated with polyvinyl butyral (PVB) to improve stability.3 IL-2 and pH sensing was achieved using gold electrodes. A pH-sensitive Al2O3 or HfO2 layer is deposited through atomic layer deposition on the pH sensor to achieve pH-sensitivity. Glucose and lactate sensing was achieved amperometrically using enzymes, glucose or lactase oxidase respectively, immobilized on a Pt electrode biased to 0.7 V. Detection could also be achieved potentiometrically by using the pH sensor to detect changes in pH caused by conversion of the enzyme product, H2O2, to H+.Figure 1 shows the capsule and sensor design. The capsule was 3D printed using a Stratasys Connex-350 and was designed to be fully insulated from solution except for an opening on the top revealing the sensors. The sensor chip is installed within the cap of the capsule which can be screwed off, but with wired connection to the electronics housed in the body of the capsule. This allows for simple replacement of the sensor chip while retaining the capsule components for reuse. Results and Conclusions Before testing within the capsule, the sensors were tested using microfluidics. Figure 2 shows the performance of the Ag/PVB pseudo-RE. The pseudo-RE shows a stable potential in different pH buffer solutions and similar sensitivity results when compared to a commercial reference electrode for pH sensing. By having a stable microfabricated pseudoRE, the sensing components will not limit miniaturization of the capsule. Figure 3 shows the response of the pH, lactate, and glucose sensors at different concentrations. The pH sensor shows high sensitivity (~53 mV/pH) over the range of pH 3 to 7. Glucose and lactate sensing was tested in 1X PBS. The glucose sensor shows a linear response to glucose concentrations from 1-6 mM with saturation at ~8 mM validating the use of the sensor within cell culture media.4 The lactate sensor shows sensitivity in the concentration range of 1-5 mM with saturation occurring around ~6 mM. The division of T cells requires aerobic glycolysis where glucose is converted to lactate. However, not all glucose is expected to be converted. The lactate concentration expected to be produced is ~1 mM.5 Selectivity towards IL-2 is achieved using anti-IL2 antibodies. Figure 4a shows the functionalization scheme for detection of IL-2. A thiol self-assembled monolayer (SAM) is attached to the gold electrode and then modified for amine conjugation using NHS/EDC. Anti-IL2 can then bind to the SAM and unbound sites are blocked with ethanolamine. Figure 4b reveals antibody attachment occurs optimally at pH 5. Surface plasmon resonance shows successful attachment of 10 ug/mL of IL-2 to the functionalized gold sensor. The next step is to demonstrate IL-2 sensing potentiometrically. The capsule’s wireless sensing capability was validated using pH sensing in a beaker. The pH sensing results in Figure 5b were obtained wirelessly from the capsule and were nearly identical to the results measured using our microfluidic system and Keithley-4200 lab instrument (Fig. 5a). This work has successfully demonstrated wireless sensing using a 3D-printed sensor capsule and functionality of electrochemical sensors for multiple CQAs for cell growth in bioreactors. These sensor capsules can enable scalability of the cell manufacturing process while ensuring delivery of an efficacious treatment. Future work will focus on capsule sensor validation in cell growth media and monitoring of cell growth in bioreactors.

Probeless non-invasive near-infrared spectroscopic bioprocess monitoring using microspectrometer technology

Real-time measurements and adjustments of critical process parameters are essential for the precise control of fermentation processes and thus for increasing both quality and yield of the desired product. However, the measurement of some crucial process parameters such as biomass, product, and product precursor concentrations usually requires time-consuming offline laboratory analysis. In this work, we demonstrate the in-line monitoring of biomass, penicillin (PEN), and phenoxyacetic acid (POX) in a Penicilliumchrysogenum fed-batch fermentation process using low-cost microspectrometer technology operating in the near-infrared (NIR). In particular, NIR reflection spectra were taken directly through the glass wall of the bioreactor, which eliminates the need for an expensive NIR immersion probe. Furthermore, the risk of contaminations in the reactor is significantly reduced, as no direct contact with the investigated medium is required. NIR spectra were acquired using two sensor modules covering the spectral ranges 1350–1650 nm and 1550–1950 nm. Based on offline reference analytics, partial least squares (PLS) regression models were established for biomass, PEN, and POX either using data from both sensors separately or jointly. The established PLS models were tested on an independent validation fed-batch experiment. Root mean squared errors of prediction (RMSEP) were 1.61 g/L, 1.66 g/L, and 0.67 g/L for biomass, PEN, and POX, respectively, which can be considered an acceptable accuracy comparable with previously published results using standard process spectrometers with immersion probes. Altogether, the presented results underpin the potential of low-cost microspectrometer technology in real-time bioprocess monitoring applications.Graphical abstract

Fast measurement of phosphates and ammonium in fermentation-like media: A feasibility study

Real-time monitoring of bioprocesses plays a key-role in modern industries, providing new information on full-scale production, thus enabling control of the process and allowing it to run at optimal conditions while minimizing waste. Monitoring of phosphates and ammonium in fermentation processes has a twofold interest: they are important nutrients for living organisms while at the same time constituting environmental nutrient pollutants, for which unnecessary use and disposal must be avoided. In this report, the possibility of simultaneous analysis of phosphates and ammonium in fermentations was verified using spectroscopy-based methods combined with chemometrics to construct calibration models. To achieve this, the models were based on synthetic samples mimicking real fermentation media, providing a dataset where the analytes were completely uncorrelated. Different at-line techniques (mid- and near- infrared spectroscopy, MIR and NIR) were evaluated for their ability to monitor quickly both analytes, in a wide range of concentrations (10−100 mM), in three media of different complexities. Partial Least Squares (PLS) models on MIR spectroscopy gave very good results, with prediction errors lower than 5 % for both analytes in all datasets. In contrast, the results for PLS models on NIR spectroscopy were inferior (prediction errors between 3 and 26 %) for both analytes, as, in the case of phosphate, it could be demonstrated that the model was based on based on indirect predictions.

Spectroscopic sensors for in-line bioprocess monitoring in research and pharmaceutical industrial application.

The use of spectroscopic sensors for bioprocess monitoring is a powerful tool within the process analytical technology (PAT) initiative of the US Food and Drug Administration. Spectroscopic sensors enable the simultaneous real-time bioprocess monitoring of various critical process parameters including biological, chemical, and physical variables during the entire biotechnological production process. This potential can be realized through the combination of spectroscopic measurements (UV/Vis spectroscopy, IR spectroscopy, fluorescence spectroscopy, and Raman spectroscopy) with multivariate data analysis to obtain relevant process information out of an enormous amount of data. This review summarizes the newest results from science and industry after the establishment of the PAT initiative and gives a critical overview of the most common in-line spectroscopic techniques. Examples are provided of the wide range of possible applications in upstream processing and downstream processing of spectroscopic sensors for real-time monitoring to optimize productivity and ensure product quality in the pharmaceutical industry.

Development of a real-time bioprocess monitoring method for docosahexaenoic acid production by Schizochytrium sp.

Oxygen uptake rate (OUR) and respiratory quotient (RQ) are key respiratory parameters for docosahexaenoic acid (DHA) production by Schizochytrium sp. HX-308 under dissolved oxygen limited conditions. To investigate the relationship of OUR and RQ with culture status, three independent cultures with different aeration rates were performed in a 50L bioreactor. OUR was found to be positively correlated with the aeration rate, which reflected the oxygen supply level in each culture. The highest biomass, reaching 124.5g/L, was achieved under the highest OUR. DHA content was found to be highly correlated with the RQ value, and the highest DHA content (44.85% in total fatty acids, w/w) was achieved in the highest RQ level, which implies that the polyketide synthase pathway was more active. OUR and RQ, which reflect the physiological state of microorganisms, are suggested as synergistic real-time bioprocess monitoring parameters for DHA fermentation.

2D fluorescence spectroscopy for real-time aggregation monitoring in upstream processing

Product aggregation is one side effect of rising yields due to process improvement and therefore accompanied with massive product loss during downstream processing (DSP). But it is already in literature described, that product aggregation also occurs during the fermentation process and is caused by various process operations [1]. Real-time bioprocess monitoring and thus on-line product quality control during upstream processing (USP) enables to address this issue during process development. For bioprocess control, 2D fluorescence spectroscopy in combination with chemometric modeling based on fluorescence signals derived from cells and medium components is a promising tool and described in literature [2]. Furthermore extrinsic fluorescence dyes are widely used to detect and quantify aggregated protein [3]. In this study, 2D fluorescence spectroscopy in combination with three different extrinsic fluorescence dyes were evaluated, in order to establish a process control tool which enables real-time product control during USP.

Synchronous fluorescence spectroscopy as a novel tool to enable PAT applications in bioprocesses

In this work, synchronous fluorescence spectroscopy (SFS) is evaluated as a new tool for real-time bioprocess monitoring of animal cell cultures. This technique presents several advantages over the traditional two-dimensional (2D) fluorometry since it provides data on various fluorescent compounds in a single spectrum, showing improved peak resolution and recording speed. Bioreactor cultures of three monoclonal antibody-producing CHO cell lines were followed in situ by both 2D and synchronous fluorometry techniques. The time profiles of the main spectral features in each data type present some differences, but principal component analysis indicated both as containing enough information to distinguish the cultures. Partial least squares regression models were then independently developed for viable cell density and antibody levels on the basis of the different fluorescence signals recorded, hiding half of the dataset for subsequent validation purposes. Regardless of the signal used, model predictions fit very well the off-line measurements; still, the synchronous spectra collected at a wavelength difference of 20 nm allowed comparable and superior performances for cell density and antibody titer, respectively, with validation accuracies higher than 91%. Therefore, SFS compares favorably with the traditional 2D approach, becoming an improved, faster option for real-time monitoring of cells and product titer over culture time. The readiness in data acquisition facilitates the design of process control strategies meeting the requirements of a PAT application.

Ex situ bioprocess monitoring techniques

Rapid development of biotechnology resulted in increasing need for bioprocess monitoring and control. Efficient process control allows optimization of process efficiency and productivity, reduction of costs and pollution, and quality control. In situ sensors for real-time bioprocess monitoring have been recently reviewed. In this paper the use of ex situ techniques for real time (or quasi real time) monitoring methods is reviewed. These include mass spectrometry, gas chromatography, methods based on semiconductor gas sensors and electronic noses for the analysis of dissolved gas and bioreactor head-space, and on-line liquid chromatography and capillary electrophoresis, spectroscopy and biosensors for monitoring of the bulk process media. Flow techniques allow for the analysis to be carried out ex situ, out of the reactor reintroducing various detection methods that are impossible to implement in an in situ set-up. Because of their versatility flow analysis techniques (continuous and segmented flow analysis, flow injection analysis, sequential injection analysis and bead injection analysis) are broadly used for sample handling and analysis of the bulk media, and their applications in bioprocess monitoring are discussed.

Rapid monitoring of virus-like particles using an optical biosensor: A feasibility study

Virus-like particles (VLPs) are multimeric proteins expressed by Saccharomyces cerevisiae. The particles are ≈80 nm in diameter and they are used as a framework for a range of biological products; for example as carriers of viral antigens. Rapid monitoring of purified VLPs was investigated using an optical biosensor. The aim was to develop an assay which may be employed for real-time bioprocess monitoring of VLPs. Problems of mass transfer of analyte were overcome through selection of a planar biosensor surface, in preference to the traditional polymer-coated surface. To prolong the surface activity for interaction analysis, a sandwich assay was developed which involved the use of a secondary capture species. It was shown that VLP concentration in pure solution could be determined within 10 min.