Innovative Solutions for Accurate Western Blot Imaging

Save time and deliver high-resolution, high-quality, and high-confidence life science images with western blotting

While the advent of X-ray film and digital imaging solutions has transformed western blot detection, successful imaging requires the combined use of an accurate protein detection technique and imaging device. The choice of imaging solution therefore depends on the protein detection method, as well as the advantages and limitations of each solution.

Key Topics in this eBook:

How to save time in Western Blotting (optimizing exposure time, automatic signal-to-noise optimization, remote image transfer, and more)
Resolve closely-spaced and multiple different protein bands on the same blot
Go beyond the immunoblot and use CCD imagers for other applications

The Amersham ImageQuant™ 800 GxP biomolecular imagers, developed by Cytiva, offer many benefits for chemiluminescence and fluorescence detection, including enhanced sensitivity, resolution, and data security. This eBook explores these solutions in detail and the advantages they offer researchers conducting western blotting in the lab.

Download the eBook to learn more about innovative imaging solutions, courtesy of Cytiva.

Innovative solutions for accurate western blot imaging

Contents

Introduction 3

Save time in western blotting 4

Optimize CCD imager signal-to-noise, without the guesswork 7

ImageQuant™ 800 for lateral flow assay development using dyes across the spectrum 14

Fluorescence reveals hidden details in flowers that attract pollinators 19

Optimizing chemiluminescence and fluorescence imaging 22

ImageQuant™ 800 for lateral flow assay development using dyes across spectrum 22

Western blot: the secrets to success in a GxP lab 27

Traceability in Western blot imaging: Considerations for GxP 31

Understanding 21 CFR Part 11 and its application in analytical labs 35

Introduction

A western blot is a common analytical technique used to detect specific proteins within a sample, evaluate the size of a particular protein and/or to determine the level of protein expression.

While the advent of X-ray film and digital imaging solutions (e.g., charge-coupled device (CCD) camera- based imagers and scanner-based systems) has transformed western blot detection, successful imaging requires the combined use of an accurate protein detection technique and imaging device. The choice of imaging solution therefore depends on the protein detection method, as well as the advantages and limitations of each solution.

The Amersham ImageQuantTM 800 GxP biomolecular imagers, developed by Cytiva, offer many benefits for chemiluminescence and fluorescence detection, including enhanced sensitivity, resolution and data security.

In this eBook, we explore these solutions in detail and the advantages they offer researchers conducting western blotting in the lab.

Science meets art:

Cytiva’s ImageQuant™ 800 imager reveals hidden detail in this image.

SAVING TIME

Save time in western blotting

How much time do you spend finding the right exposure settings for Western blots? Find out how the right CCD imager can help you work more efficiently, freeing valuable resources to drive your research forward, faster.

Western blotting efficiency in the lab

Using time in the lab efficiently promotes success in research with faster generation of key data leading to scientific breakthroughs. To drive efficiency, it is important

to watch for ways to improve and refine routine practices and procedures, as time savings in these daily repetitive activities can add up quickly.

Western blotting is an affordable process that reliably answers important basic questions regarding proteins of interest. For this reason, Western blots, or protein immunoblots, are used daily by most labs working on protein-based research.

While the Western blotting method has stayed largely unchanged over the last few decades, there remains opportunity to increase efficiency in the process. Saving even just a little bit of time on every Western blot can add up to substantial time-savings over the course of a week, month, or year—time you and your colleagues can spend on other valuable research efforts.

Optimizing exposure times in CCD imaging

Western blots often have bands of varying intensity, with low-expression proteins presenting with lower band intensity and highly expressed proteins represented by more intense bands. Low-intensity bands need longer exposure times to be visible, while the high-expression bands require shorter exposure times to avoid saturation.

Cytiva

The varying imaging requirements of low- and high-expression proteins make visualizing both on the same blot a challenge. Although the introduction of digital imagers has provided substantial improvements over X-ray films, the dynamic range of many CCD imagers today remains limited. The user has to choose the optimal exposure settings by trial-and-error, and it is often not possible to visualize both low- and high-intensity bands on the same blot without some trade-off.

Another challenge of this trial-and-error approach is the race against time to find the optimal exposure conditions, usually requiring multiple test exposures to get the best possible image. As the chemiluminescent reaction progresses, the signal-to-noise ratio (SNR) peaks and begins to drop until the signal is indistinguishable from noise. There is also a risk that membranes dry out over time.

Once the chemiluminescent reaction progresses and the signal becomes weaker or undetectable, it might be necessary to redo the blot if you have not yet been able to find optimal exposure conditions. If you do have an image, the SNR might limit your ability to quantitate across strong and weak bands simultaneously.

Some CCD imagers offer a function that adjusts exposure time automatically; however, this function is based on the brightest signal on the blot and cannot improve the SNR of the images. This approach compromises the visibility of lower intensity bands, which are possibly the bands you are most interested in seeing.

SNOW mode saves time with automatic signal-to-noise optimization

The key to saving precious time when using Western blots for protein analysis is capturing an image with optimal SNR quickly and reliably. Our proprietary intelligent algorithm, the SNOW (signal-to-noise optimization watch) imaging mode, can help you detect weak bands without saturating strong bands and achieve high sensitivity without compromising image quality.

SNOW imaging is available with the Amersham ImageQuantTM 800 GxP biomolecular imager (Fig 1). When using this imaging mode, the system continually checks and averages image intensity data from multiple exposures

to automatically optimize the Western blot image. The imaging algorithm stops the process once the SNR peaks to acquire the best possible image. This method eliminates the need to spend time optimizing exposure and capture settings and stretches the dynamic range without compromising sensitivity and resolution.

If you need to quantitatively analyze both high signal and weak bands, trust the SNOW imaging mode to automatically optimize exposure time and maximize SNR; the output is a single image file, ready for analysis.

Fig 1 . The Amersham ImageQuantTM 800 GxP biomolecular imager helps save time in Western blot imaging.

Remote Image transfer and scheduling

Easy-to-use software on the Amersham ImageQuantTM 800 GxP biomolecular imager can help improve research efficiency by enabling remote system management, saving you a trip to the lab. The ImageQuantTM CONNECT software allows you to schedule equipment usage and access image files from your desk (Figs 2, 3, and 4).

The Amersham ImageQuantTM 800 GxP biomolecular imager software includes automatic color marker overlay, saving time by automatically imaging and overlaying white light color marker images with chemiluminescence images of the same blot.

This approach makes it easier to determine the molecular weight of your bands of interest. With most other imagers, users have to take separate white light and

chemiluminescent images and manually overlay them in a separate analysis software. The Amersham ImageQuantTM 800 GxP biomolecular imager also automatically provides users with both grayscale and colored images.

High quality, efficient CCD imaging

Small time savings in tasks that are part or many researchers’ daily routine lead to improvements in overall research productivity over time.

Designed with features such as SNOW imaging and ImageQuantTM CONNECT software, the Amersham ImageQuantTM 800 GxP biomolecular imager generates the best possible images with improved data handling to deliver the ultimate solution for fast, efficient Western blotting.

Fig 2 . Use ImageQuantTM CONNECT software from your office to access images and the scheduler tool on board the instrument.

Fig 3 . The ImageQuantTM CONNECT tool can be used to view the status of all the Amersham ImageQuantTM 800

GxP biomolecular imagers connected to the same local network in your facility.

This capability allows you to choose an available instrument and plan your experiments.

Fig 4 . Perfect for a busy multi-user lab environment, the on-board scheduler application can be used to block time on the instrument to plan and run

your experiment. Easily access the scheduler from your office via the remote ImageQuantTM CONNECT software to view and manage bookings.

Optimize CCD imager signal-to-noise, without the guesswork

Delivering high-resolution, high-quality, and high-confidence life science images, beyond Western blots.

Many laboratory CCD imaging systems have automatic exposure modes. For Western blots, these modes attempt to find an exposure time that is a favorable compromise between saturating the more intense bands and underexposing the weaker bands on a blot. As a result, the dynamic range is limited, and it is challenging to quantitate low intensity bands in the presence of high intensity bands. This difficulty is seen when a target protein is in the presence of a highly expressed protein.

The ImageQuantTM 800 system uses the proprietary intelligent SNOW imaging algorithm to automatically find the optimal signal-to-noise ratio (S/N) with minimal input or guesswork from the user. This approach provides the sensitivity needed

to detect faint bands that could not be visualized by conventional imaging without saturating stronger signals.

The SNOW imaging mode process involves the following steps:

A pre-capture exposure to identify an optimum exposure time
Selection of the region of interest and background
Multiple automatic exposures, continuously averaged to find the optimal S/N

The SNOW imaging mode is distinct from other auto-exposure modes in that it works by capturing multiple images and continuously averaging the signal. This image averaging process effectively minimizes random noise, thereby improving the signal- to-noise ratio (Fig 1).

Fig1 . Image capture and averaging by SNOW algorithm reduces noise to maximize S/N.

(A) Line profiles from continuously averaged 7.5 s exposures with SNOW algorithm, from first capture to the average after capture 73, shown by (B) a reduction in noise and stable signal and resulting in (C) an increase in S/N.

The only user input required for the SNOW imaging process is selection of background and region of interest during the initial pre-capture. The user can then watchthe signal-to-noise ratio improve in real time for their region of interest until the system reaches an optimum S/N. Figure 2 demonstrates how the SNOW process reduces background noise and improves the S/N.

Fig 2 . ImageQuantTM 800 system control software and SNOW exposure mode in progress. The image of a dilution series for a CyTM5-labeled antibody transferred to a Western blot membrane was continuously updated during the averaging process. In this image, the signal-to-noise ratio is close to its maximum value. The SNOW mode subsequently stopped automatically when the S/N started to decrease, and the software saved the image with maximum S/N.

Fig 3 . Comparison of conventional and SNOW imaging approaches for Western blots in chemiluminescence mode. (A and B) Conventional imaging with short exposures. (C) 93 s SNOW detection mode run on ImageQuant 800 CCD imager, consisting of multiple short exposures with image averaging to reduce noise. (D) Single 93 s exposure without the use of the SNOW algorithm. Compared to conventional imaging approaches, SNOW detection mode results in

a broader linear dynamic range and minimal background noise, enabling the visualization and quantitation of protein bands from a wider range of signal intensities without saturation.

The SNOW algorithm removes the guesswork from the conventional trial-and-error approach to finding optimal exposure times. This automated mode improves the efficiency and productivity of Western blot or DNA gel workflows by increasing the chance that all bands of interest will be within the broad linear dynamic range and removing the need to repeat experiments or develop multiple blots and DNA gels. Figure 3 compares different exposures for imaging a Western blot, showing the various effects of these approaches on image background noise.

Achieving this level of S/N optimization requires not just the SNOW algorithm, but also robust optics and hardware in the form a high-resolution, 8.3-megapixel CCD camera with a large aperture F 0.74 Fujifilm™ lens. The ImageQuant™ 800 system represents the culmination of 10 years of ongoing partnership, bringing Fujifilm’s expertise in optics together with our expertise in life science imaging.

See deeper into protein levels with Western blot analyses

Another common challenge with both digital CCD imaging and X-ray film is the ability to resolve closely-spaced and multiple different protein bands on the same blot. This challenge would typically require the following strategies to resolve:

An extended polyacrylamide gel electrophoresis (PAGE) run in the hope that closely-spaced bands separate sufficiently for resolution
Duplicate Western blots
Stripping and reprobing with additional primary antibodies
For a total protein stain, distinguishing bands on a Coomassie stained gel can be challenging; however, epi- and trans-white illumination combined with the high- resolution camera of the ImageQuant™ 800 CCD imager provides the ability to resolve proteins of similar molecular weights with as little as 0.5 mm spacing (Fig 4A).
Fig 4 . Differentiating between closely-spaced bands and multiplexed imaging of different proteins by fluorescence on an ImageQuantTM 800 CCD imager. (A) High-resolution colorimetric imaging of Coomassie stained gel demonstrates the ability to resolve bands on a gel 0.5 mm apart. (B) Three-color multiplexed overlay image of Western blot nitrocellulose membrane. Target ERK proteins detected using IR long (red) LED-filter combination and GAPDH detected using IR short (green) on the same blot.
For targeting multiple proteins, fluorescent Western blots provide a straightforward alternative to chemiluminescence. Supported by IR short and IR long illumination capabilities, the ImageQuant™ 800 system enables multiplexed detection of several proteins on the same blot (Fig 4B).
Taking CCD imagers beyond the immunoblot
Analysis of Western blots is the primary application of CCD imagers in many laboratories. While some CCD systems make it possible to image for other applications, these imagers might not have been designed with these applications in mind. This accommodation could require changes to hardware and settings that complicate workflows. A compromise in image quality or sensitivity is also possible.
By comparison, the ImageQuant™ 800 CCD imager simplifies and delivers high- quality images for a broad range of applications with flexibility in illumination and versatility in allowed sample type built in. The light modes, for example, include:
Epi-white (470 nm to 656 nm)
Epi-UV (360 nm)
Epi-RBG (635 nm, 460 nm, and 535 nm)
Epi-IR short (660 nm)
Epi-IR long (775 nm)
Trans-White

These illumination options, as well as a variety of customizable filters, enable users to image a variety of samples across the full spectrum using a single instrument. This flexibility allows users to complete a range of useful research functions and applications while saving space and simplifying workflows.

Colony counting and analysis

Counting cell colonies can be tedious and time consuming. A colony counting system takes up bench space while providing one very specific function. Additionally,

reproducing the same image and analysis across different petri dishes with a standard hand-held camera can present a challenge.

The ImageQuant™ 800 CCD imager software takes advantage of illumination flexibility, enabling automatic colony analysis using the optical density (OD), fluorescence, or UV modes (Fig 5). An additional non-parallax (NP) lens accessory allows chemiluminescence imaging to be used for petri dishes and multi-well plates without introducing optical artifacts.

Fig 5 . Colony imaging options for petri dishes with the ImageQuantTM 800 system. (A) Optical density measurements providing a direct measurement of the OD of each colony. (B) Full- color imaging. (C) Epi-UV fluorescence imaging to capture the auto-fluorescence of the cells. Chemiluminescence imaging is also possible using the NP lens accessory tray.

Host cell protein analysis

Host cell protein (HCP) levels provide a purity indicator for biologics, enabling manufacturers to evaluate their purification strategies and meet pharmacopeia recommendations. HCP analysis represents a critical step in gaining regulatory approval.

Analyzing an HCP ELISA usually requires a separate microplate spectrophotometer. With ImageQuant™ TL analysis software on the ImageQuant™ 800 system, however, HCP ELISAs can be analyzed without the need for a dedicated plate reader (Fig 6).

Fig 6 . Quick evaluation of HCP ELISA using the HCPQuant ELISA kit and the ImageQuantTM 800 CCD imager. (A) White-light image shows yellow color change upon detection of HCP proteins from Chinese hamster ovary (CHO) cells. (B) Array analysis image where suitable reference samples were available, and (C) the standard curve generated by software for quantitation.

As an extension of its chemiluminescent and fluorescent Western blot imaging modes, the ImageQuant™ 800 CCD imager provides straightforward imaging of HCP coverage assays. These assays can be from a traditional Western blot, differential in- blot electrophoresis (DIBE), or difference in-gel electrophoresis (DIGE) approach.

DNA gel visualization and macroscopic imaging applications

The conventional method of imaging a DNA gel uses a dedicated UV platform with a connected computer, camera, and printer. The UV illumination mode on the ImageQuant™ 800 removes the need for a separate device and generates an image file that is easily accessible locally and remotely using ImageQuant CONNECT software.

The versatility of the ImageQuant™ 800 system continues beyond laboratory gels, blots, and plates. The UV illumination function, for example, has been used to study structures at the macroscopic level, such as vein-like patterning on flowers revealed under fluorescence excitation (Fig 7).1

Fig 7 . Dendrobium nobile orchid imaged in the ImageQuantTM 800 system using different LED and filter combinations. The imager revealed how parts of the flower fluoresced under different excitation wavelengths, showing vein-like patterns.

Getting the most out of a CCD imager

Combining multiple imaging instruments into one CCD imaging system might raise concerns about instrument availability, especially in multi-user laboratories. If an instrument is unavailable or in high demand, there is risk of restricting researchers and creating bottlenecks relating to instrument access.

The ImageQuant™ 800 CCD imager and associated ImageQuant™ CONNECT software are designed with these challenges in mind. Connected to the local network, the system software enables both local and remote scheduling, as well as access to images from previous runs. This accessibility eliminates the need to physically transfer images with a USB drive.

CCD imagers can grow with a lab’s research needs

A CCD imager is a substantial investment for any lab or research facility. It can be challenging to strike the optimum balance between meeting a laboratory’s ever- changing research needs and avoiding redundancy in equipment.

While the ImageQuant™ 800 biomolecular imager provides a considerable array of functionality, laboratories that do not require the full suite of options can still benefit from the high-resolution, high-quality, and high-confidence images provided. The system is available in four configurations: ImageQuant™ 800, ImageQuant™ 800

UV, ImageQuant™ 800 OD, and ImageQuant™ 800 Fluor. Applications and light sources for each configuration are described in Table 1. Each option provides a straightforward upgrade path to expand and adapt to accommodate the unique and changing needs of a research group over time.

Table 1: ImageQuantTM 800 system configurations and applications.

Applications	Light Sources	ImageQuantTM 800	ImageQuantTM 800 UV	ImageQuantTM 800 OD	ImageQuantTM 800 Fluor
Chemiluminescence with color marker overlay	n/a	√	√	√	√
Gel documentation	Epi-white	√	√	√	√
Stained gels	Trans-white		√	√	√
Optical density (OD) measurements	Epi-RGB			√	√
RGB fluorescence imaging of blots	Trans-white				√
IR short and IR long fluorescence imaging of blots	Epi-IR short, Epi-IR long				√

We provide a range of solutions for life science imaging applications and workflows. Through a combination of robust hardware and innovative software, including the unique SNOW imaging mode, the ImageQuantTM 800 CCD imager enables flexible and versatile imaging across multiple applications. To learn more about the SNOW imaging algorithm or for support with any other aspect of the imaging workflow, contact our Scientific Support team.

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One system, many uses

Overview Amersham ImageQuant 800 TM biomolecular imager

The Amersham ImageQuant™ 800 GxP biomolecular imager provides top quality gel and blot images for laboratory analysis, along with full control of your data through automation.

In combination with analysis software, ImageQuant™ 800 GxP supports your compliance with 21 CFR Part 11 and EU GMP Annex 11 frameworks. Watch this short video to learn more about how this imager is designed specifically for regulated environments where data traceability, integrity, and accountability are critical.

Watch video

ImageQuant™ 800 for lateral flow assay development using dyes across the spectrum

Development of assays based on lateral flow has led to low cost and effective large-scale screening of infectious diseases with high specificity. Lateral flow assays consist of membranes, antibodies, and reagents for detection of a color change or fluorescent dye (Fig 1). Lateral flow assays are an area of intense research and have led to highly robust and sensitive products, including a range of Whatman™ diagnostic solutions from Cytiva. In this article we investigate the use of Conjugated Polymer Nanoparticles (CPNs™) and Amersham ImageQuant™ 800 systems for lateral flow assays and Western Blotting.

ImageQuantTM 800 GxP biomolecular imager

During assay development it is important to optimize the detection step, either based on color or fluorescence detection. ImageQuant™ 800 is a CCD based imaging system for colorimetric and fluorescence imaging. It is equipped with six different types of LED lights and

filters so users can image across the full spectrum. It also provides white light colorimetric imaging, both in epi- and transillumination mode. ImageQuant™ 800 allows the user to make any custom LED light and filter combinations for

imaging of various dyes with different excitation and emission wavelengths including UV, RGB, and IR long and short wavelengths. Furthermore,

the ImageQuant™ 800 can also accommodate custom filters from third parties making it ideal for any assay development work.

Fig 2 . The ImageQuantTM 800 imaging system comes equipped with touchscreen, two tray positions, and an easy-access filter door.

Fig 1 .. Lateral flow assay architechture

Fig 3 . Image across the full spectrum with the different LED light sources of the ImageQuantTM 800 Fluor imager.

Conjugated Polymer Nanoparticles (CPNs™)

Fig 4 . Fluorescence emission spectra of CPNs™ from Stream Bio’s (spanning 420 nm to 1000 nm). CPNs™ fluorescence at specific wavelengths is due to the chemical composition. The excitation and emission characteristics of the CPNs™ are tailored by selecting specific conjugated polymers for the core.

Stream Bio CPNs™ are a new class of molecular bioimaging probes. The polymer material provides a high degree of photostability and highly specific targeting. CPNs™ together with a sensitive and flexible imaging system, such as the ImageQuant™ 800, provide the high sensitivity needed for a range of life science applications, including western blot, lateral flow rapid diagnostic tests and ELISAs.

CPNs™ in lateral flow assays for a strong, consistent signal

CPNs™ exhibit many useful characteristics for lateral flow assay development, including:

Fig 5 . CPN610 or europium was used as detection agents by linking to anti-hCG detection antibodies. The strips were imaged using the ImageQuant™ 800 and the signal from the test and control lines analyzed to give a mean pixel intensity for each line. The image was captured using the Blue epi (460 nm) and Cy™3 UV filter for the CPN610 signal and UV epi (365 nm)

and Cy™3 UV filter for the europium signal. White grayscale was used to indicate fluorescence signal, i.e., a high signal is shown as white in the image.

Fluorescence emission across a wide range of wavelengths from 420 to 1000 nm, with possible multiplexing
Bright and robust, with resistance to photobleaching
Contain iron oxide and can be moved using magnetic fields
Conjugated with well known surface chemistries, such as streptavidin-biotin, thiol-amine coupling, and “click” chemistry

Lateral flow strips for the detection of human Chorionic Gonadotropin (hCG) were tested in triplicate using concentration of hCG from 100 μg/mL to 1 ng/mL (Fig 5). The data from the test lines on the lateral flow strips were then plotted against the hCG concentration (Fig 6).

Fig 6 . Multiple replicates show consistent strong signal from CPNs™ with higher sensitivity than Europium.

Detection of COVID spike proteins using CPNs™ and ImageQuant™ 800 imager

CPN510B was used to detect spike protein targeting domain and to define the limit of detection for a COVID diagnostic assay. The full-length spike protein and its S1 and Receptor Binding Domains (RBD) of the virus SARSCoV2

(causative agent of COVID-19) were spotted onto a nitrocellulose membrane (50 ng per spot). They were then probed with CPN510B covalently linked to an anti- spike protein molecularly imprinted polymer and imaged using the ImageQuant™ 800 (Fig 7). Strong signals were obtained from the full-length spike protein and S1 domain with the RBD giving a weaker signal.

The full-length spike protein from the SARS-CoV2 virus was then spotted onto a nitrocellulose membrane (Fig 8). The spots were then probed with CPN510B

Fig 7 . Nitrocellulose membrane spotted with 50 ng of full-length spike protein,

S1, and RBD of the virus SARS-CoV2. The image was captured using the UV epi (365 nm) and Cy™2 (525BP20) filter for the CPN510B signal.

Fig 8 . Nitrocellulose membrane spotted with varying concentrations of full-length spike protein from the SARS-CoV2 virus. The image was captured using the UV epi (365 nm) and Cy™2 filter for the CPN510B signal.

covalently linked to an anti-spike protein molecularly imprinted polymer and imaged using the ImageQuant™ 800 imager. Signals could be detected at each concentration including 0.5 ng.

Detection in the IR region using ImageQuant™ 800 imager

CPN™ performance in infrared regions was evaluated using ImageQuant™ 800. Lateral flow strips were tested with infrared emitting CPNs™ and imaged using various filter settings on the ImageQuant™ 800 (Fig 9). Fluorescent material has the characteristic of absorbing light at some wavelengths (excitation) and

emitting light at other wavelength. With the combination of filters available on the ImageQuant™ 800 it is possible to see both the fluorescent signal (white line) and an absorbance signal (black line) against the grey background of the nitrocellulose strip. ImageQuant™ 800 filter sets can be selected to match the emission properties of the CPNs, such as CPN840 (ex/em =650/840 nm).

Fig 10 . Mouse-anti-CD44 (500 ng 1 μg) was run on a PAGE gel and transferred to a nitrocellulose membrane. The membrane was probed with CPN840

linked to anti-mouse IgG which selectively bound, giving a bright signal at the expected size (150 kDa). The image was captured using the 660 nm IR short epi and 46 nm IR long (836BP) filter for the CPN840 signal. White grayscale was used to indicate fluorescence signal, i.e., a high signal is shown as white in the image.

CPNs™ and ImageQuant™ 800 imager for Western Blotting

The increase in sensitivity achievable with CPNs™ allow them to be used to probe Western blots linked directly to the primary antibody rather than using a labelled secondary antibody (Fig 11). This avoids incubation with the secondary antibody leading to significant time savings.

Fig 9 . Lateral flow strips were run using a range of infrared CPNs™ as detection agents and the strips were imaged using four different filter sets on the ImageQuant™ 800. White grayscale was used to indicate fluorescence signal, i.e., a high signal is shown as white in the image.i.e. a high signal is shown as white in the image.

Fig 11 . Nitrocellulose membrane was spotted with human chorionic gonadotropin (hCG) at 1 mg, 500 ng, and 250 ng. It was then probed with CPN610 linked to anti-hCG antibody or

probed with a mouse anti-hCG antibody followed by QDot 565 linked to anti-mouse antibody. Signals at each amount of hCG were comparable. The image was captured using the UV epi (360 nm) excitation source and Cy™3 40 nm UV emission filter.

Conclusion

Assays using lateral flow methods have led to lower cost methods for large-scale screening of infectious diseases with high specificity. In this article we have shown several applications use of CPNs™ on the ImageQuant™ 800 for lateral flow assays and Western Blotting. ImageQuant™ 800 GxP biomolecular imagers are ideal

for evaluating sensitivity, dynamic range, and limits of detection for use of such fluorophores in lateral flow diagnostic assay development.

About Cytiva’s diagnostic assay development services

Cytiva Diagnostic Services helps you accelerate diagnostic assay development, from early-stage concept and prototype to manufacturing and commercialization. Whether creating a custom magnetic bead, a specialized enzyme, or a diagnostic kit, our team in Cardiff, UK offers custom solutions including lyophilization, conjugation, contract manufacturing, custom biology and custom plates. Our team in Dassel, Germany builds on our Whatman heritage of quality and innovation, providing access to our expertise, hands-on training, infrastructure and proven development methodologies for rapid diagnostic test design. Find out more.

About Stream Bio

StreamBio develops and manufactures a range of bioimaging molecular probes that cover the visible and near infra-red spectrum: Conjugated Polymer Nanoparticles (CPNs™), for applications in R&D, in vitro diagnostic assays and therapeutic research. Invented in the research labs of King’s College London,

Stream Bio was founded as the vehicle through which to demonstrate the wide- ranging benefits and applications of CPNs™. Stream Bio’s technology is already making a positive impact on in vivo R&D, diagnostics and therapeutics.

Cytiva Protein Research Series

Launch series

Watch this video to learn how to overcome the

data analysis bottleneck in drug discovery and why data transparency and transparent AI are critical for the analysis of biophysical data such as surface plasmon resonance (SPR).

UTILIZING FLUORESCENCE IMAGING

Fluorescence reveals hidden details in flowers that attract pollinators

Color patterns and color contrasts are the most recognizable way for flowers to attract pollinators like birds and bees. In addition, certain areas of the flower including some pollen and nectar are fluorescent as well, which could potentially aid in the pollination of flowers (1–6).

Using multiplex fluorescence imaging we found intricate and detailed fluorescence patterns in flowers which were not detected by visual color inspection. In particular, the central parts exhibit strikingly different fluorescence properties when illuminated

with Ultraviolet (UV), visible, and Infrared (IR) light. The potential role of fluorescence

in flower signaling and communication is an exciting new field of research [1–6] which requires state of the art imagers. The Amersham ImageQuant™ 800 GxP biomolecular imager is an ideal companion with its wide range of narrow range light-emitting diodes (LED) and multiple emission filters, which can be combined freely. The illumination range spans from UV to IR.

The following flowers were imaged using Amersham ImageQuant™ 800 GxP biomolecular imager and images were analyzed using ImageQuant™ TL 10.1 analysis software.

Science meets art:

Cytiva’s ImageQuantTM 800 imager delivers flexible, high resolution imaging across the full spectrum from UV to infrared.

5 mm

Fig 1 . A daisy (Bellis perennis) was imaged with ImageQuant™ 800 using (A) colorimetric and

(B) fluorescent imaging modes at UV (magenta), Cy™2 (yellow), Cy™3 (green), and IR long (blue).

Fig 2 . A siberian squill (Scilla siberica) was imaged with ImageQuant™ 800 using (A) colorimetric and (B) fluorescent imaging modes at UV (red), Cy™3 (yellow), and Cy™5 (green).

5 mm

Fig 3 . A cowslip (Primula veris) was imaged with ImageQuant™ 800 using (A) colorimetric and (B) fluorescent imaging at Cy™2 (red), Cy™3 (magenta), and IR long (yellow).

Fig 4 . A dandelion (Taraxacum officinale) was imaged with ImageQuant™ 800 using (A) colorimetric and (B) fluorescent imaging at UV (cyan), Cy™2 (red), Cy™3 (yellow), and IR long (blue).

5 mm

Fig 5 . A yellow wood anemone (Anemone ranunculoides) was imaged with ImageQuant™ 800 using (A) colorimetric and (B) fluorescent imaging modes at UV (red), Cy™3 (green), Cy™5 (yellow), and IR long (blue).

Fig 7 . The Amersham ImageQuant™ 800 GxP biomolecular imager allows high resolution imaging across the entire visible spectrum, from UV to infrared. The daisy Bellis Perennis contains multiple yellow disc florets and white ray florets. The open disc florets display a characteristic fluorescence emission observed with UV, Cy™2, and Cy™3 LED-filter settings compared to the budding florets in the center. In the fake color overlays, these open florets appear as pink dots, approximately 0.5 mm wide. The Amersham ImageQuant™ 800 1×1 binning allows such sub-mm details to be clearly resolved.

Fig 6 . A wood anemone (Anemone nemorosa) was imaged with ImageQuant™ 800 using (A) colorimetric and (B) fluorescent imaging at UV (green), Cy™3 (blue), and IR short (yellow).

References

O. Ostroverkhova, G. Galindo, C. Lande, J. Kirby, M. Scherr, G. Hoffman and S. Rao, “Understanding innate preferences of wild bee species: responses to wavelength-dependent selective excitation of blue and green photoreceptor types,” Journal of Comparative Physiology A, vol. 204, p. 667–675, 2018.
F. Gandía-Herrero, F. García-Carmona and J. Escribano,“Floral fluorescence effect.,” Nature 437,
p. 334, 2005.
C. Van der kooi, A. Dyer, P. Kevan and K. Lunau, “Functional significance of the optical properties of flowers for visual signalling.,” Annals of Botany 123, p.263–276, 2019.
A. Iriel and M. Lagorio, “Is the flower fluorescence relevant in biocommunication?,”
Naturwissenschaften 97, p. 915–924, 2010.
K. Lunau, Z. Ren, X. Fan, J. Trunschke, G. Pyke and H. Wang, “Nectar mimicry: a new phenomenon,” Scientific Reports, p. 7039, 2020.
S. Mori, H. Fukui, M. Oishi, M. Sakuma, M. Kawakami, J. Tsukioka, K. Goto and N. Hirai, “Biocommunication between Plants and Pollinating Insects through Fluorescence of Pollen and Anthers,” Journal of Chemical Ecology, vol. 44, p. 591–600, 2018.

Optimizing chemiluminescence and fluorescence imaging

Extend dynamic range and avoid saturation with SNOW detection

The fine balance of photon collection in image capture

The art of capturing biomolecular images is, at its heart, about harvesting photons. Collecting more photons from a sample results in sharper images and detection of faint protein bands which otherwise could have been missed. More photons also leads to a greater signal-to-noise (S/N) ratio and increased quantitation confidence.

The present generation of ECL reagents for chemiluminescence-based detection, such as ECL Prime, uses the enzymatic horseradish peroxidase (HRP) luminol reaction to deliver a stable, high signal. In fluorescence-based detection, CyDyeTM fluorophores are the industry standard and now span across the entire visible range, with Amersham CyDye TM 700 and 800 near infrared (NIR)-labeled antibodies recently taking center stage. Genomic sequencing companies now offer both public and private NGS services for genetic testing. To remain cost-efficient and cost- effective, these companies need to maintain a high sample throughput, processing large numbers of NGS samples per day and keeping their sequencing systems busy churning out data.

Modern imagers have light sources, emission filters, and lenses designed to maximize detection of emitted sample photons. If more detected photons are better, when in the imaging process should we stop collecting photons and how can we decide the best exposure time? Image capture is usually allowed to continue to obtain maximum signal, with the limiting factor being saturation of bands of interest.

Fig 1 . There is a wide range of fluorescence excitation and emission options available on the Amersham ImageQuantTM 800 GxP biomolecular imager.

Grayscale images in .TIF format, composed exclusively of gray shades or levels, are used for quantitation in applications like Western blotting. In a 16-bit .TIF file generated by the imager, there are 65 535 gray levels. When surplus photons are collected, the maximum image pixel value remains at 65 535, regardless of the exposure time, and results in saturation. Yet, we might still want to collect more

Fig 2 . Grayscale .TIF files are composed of different levels of gray shades. 16-bit files have a higher number of grayscale levels than 8-bit files.

photons to see details in the images and detect weak bands. So, how can we overcome this challenge?

SNOW detection for image optimization

The Amersham ImageQuant TM GxP biomolecular imager introduces a novel capture mode called SNOW (named for Signal-to-Noise Optimization Watch) which allows for the collection of photons for longer exposure times without saturation. The technology is based on recording each image after an appropriate time to avoid saturation, and then averaging all recorded images into a final image. Image details are more visible and linear dynamic range is extended, avoiding saturation.

SNOW imaging mode also allows users to view image quality improvement in real time and auto-stops when the best image with the highest signal-to-noise ratio has been achieved. This capability eliminates the trial and error of image capturing and removes the uncertainty that comes with selecting one image from many for analysis.

Simply start the automatic SNOW imaging mode on the Amersham ImageQuantTM 800 GxP biomolecular imager and come back to view the final optimized image.

In this article, users of the Amersham ImageQuantTM 800 CCD GxP biomolecular imagers can find information on how to leverage SNOW detection to capture the best images. The SNOW detection mode can be used with both ECL and fluorescence detection of proteins on a Western blot and for any binning setting, permitting image capture of even the most demanding samples for which high resolution is critical.

Two-fold S/N improvement in chemiluminescence detection

Amersham ECL TM Prime is a highly sensitive chemiluminescent detection reagent characterized by extremely stable signal emission. Combined with SNOW detection mode, this reagent enables longer exposure times and higher signal-to-noise ratios, resulting in better band resolution.

Fig 3 . Amersham ImageQuantTM 800 GxP biomolecular imager with SNOW imaging mode. Fig 4 . Images of the same membrane from auto mode (7 min, first) versus SNOW mode (49

min, second) using ECL Prime. The signal from the main band is approximately 30 000 counts for both images. The limit-of-detection band had twice the S/N ratio for the SNOW capture.

Amersham

Fig 5 . Example of signal-to-noise improvement during SNOW capture using ECL Prime.

Fig 7 . Excitation and emission spectra of Amersham CyDyeTM 700 secondary antibodies.

Over three-fold S/N improvement in fluorescence detection Achieve exceptional linearity even while detecting

Amersham Protran Premium 0.45 µm NC Goat anti-mouse CyTM3 (Cytiva), 1:2500

Goat anti-rabbit CyTM DyeTM 700 (Cytiva), 1:15 000

weak signals

Fig 6 . Auto versus SNOW with CyTM3 detection of GAPDH using Amersham ECLTM Plex CyTM 3. The S/N of the weakest detectable band was improved by 3.5 times, from hard to observe (auto) to easy to quantitate (SNOW).

Fig 8 . Auto (1 s) versus SNOW (27 s) of a calibrated light source which emits 1 pW, 10 pW, 100 pW, and 1000 pW. Both images show excellent linearity (k = 1.01) across the dynamic range and the SNOW image exhibits almost four times lower noise levels compared to the auto image.

UV (blue) + CyTM3 (red) + CyTM5 (green)

R+G+B Visible IRlong (blue) + CyTM2 (red) + IRshort (green)

Noise reduction to detect weak protein signals High resolution imaging across the entire spectrum

Fig 9 . Amersham host cell protein (HCP) DIGE membrane with CHO lysate (50 μg) and CyTM3 detection of K1 control protein (1:200 dilution). The auto image was captured in 1.5 s and the SNOW image in 2 min 29 s, both with 1×1 binning. The noise level is markedly improved with SNOW, which allows detection of weak bands with greater confidence.

Reduce noise and detect strong and weak signals without saturation

Fig 10 . An auto IR-short (0.1 s) image (A1, A2) compared to a SNOW captured (17.7 s) image (B1, B2). The contrast of the A1 and B1 images was set to view the strong signal of the central flowers, and the contrast of the A2 and B2 images was set to view the white florets. The extended dynamic range allows both the weak and strong signals to be captured in the same image with SNOW.

Fig 11 . The Amersham ImageQuantTM 800 GxP biomolecular imager allows high resolution imaging across the entire visible spectrum, from UV to infrared. The daisy Bellis Perennis contains multiple yellow disc florets and white ray florets. The open disc florets display a characteristic fluorescence emission observed with UV, CyTM2, and CyTM3 LED-filter settings compared to the budding florets in the center. In the fake color overlays, these open florets appear as pink dots, approximately 0.5 mm wide. The Amersham ImageQuantTM 800 GxP biomolecular imager 1×1 binning allows such sub-mm details to be clearly resolved.

Conclusion

When combined with detection reagents that offer long signal stability in chemiluminescence, such as Amersham ECL TM Prime, the SNOW detection mode can be invaluable in detecting weak signals without saturation. In fluorescence detection mode, this novel setting also helps achieve high sensitivity and high resolution without saturation. SNOW detection on the Amersham ImageQuant TM 800 GxP biomolecular imager increases signal-to-noise ratio and improves image quality for accurate quantitation in biomolecular research.

Watch video

Watch this short, animated video to learn more about Cytiva’s

Amersham ImageQuant™ 800 GxP biomolecular imager.

This system is sensitive, flexible and designed to support FDA 21 CFR Part 11 EU GMP Annex 11 regulations.

A novel approach to biomolecular

imaging

Western blot: the secrets to success in a GxP lab

See how high-quality imaging with sensitivity, resolution, and data security ensures confidence in your biomolecular research

If you’re developing a new drug, cosmetic, or food product, you’re probably familiar

with GxP. GxP – or good practice – guidelines define and track each stage of development and manufacturing to ensure that your final product not only performs its intended purpose, but that it’s also safe for consumer use . Having a GxP framework in place is becoming increasingly important – and data traceability, accountability, and integrity are at the heart of this .

“GxP is so very important today, especially for our customers in pharma and biopharma,” explains Sowmya Balachandran, Global Product Manager at Cytiva.

“Good traceable data not only increases confidence in your final product but also helps you track and identify any issues should they ever arise.”

Unchanged for the past four decades, western blotting is a very simple, high-impact tool that helps answer some very basic but important research questions: Are there proteins present here? What proteins are present? How much of my target protein is present? As a result, western blotting has had a lasting consequence on workflows across research fields.

“It’s one of those underrated techniques because it’s perhaps not the most exciting thing around to talk about, but it’s one of those techniques without which we wouldn’t have the answers that we have today,” says Sowmya.

Despite its simplicity, gel electrophoresis and western blotting is a somewhat fragmented process. From the initial sample preparation to final image analysis, each step requires different instruments, consumables, and reagents that often come from different suppliers.

This presents many challenges. Firstly, the success of a western blot depends on the user’s level of experience. If you can master western blotting as a skill, then you can get good results easily and cheaply – which is partly why many scientists rely heavily on this method. Then, after you’ve prepared a good blot, the next hurdle is making sure that you have access to an imaging system that can adequately detect target proteins for reliable downstream analysis.

“Anyone who’s working on proteins will perform western blots and need to image them, so to be able to provide a product that almost every research lab can use feels amazing,” says Sowmya.

Sowmya Balachandran, Global Product Manager at Cytiva works to develop new imaging solutions & support products for use in biomolecular research

In this interview, Sowmya shares insight into the advancement of this fundamental technique for GxP-certified labs. She

Sowmya Balachandran’s top tips for achieving GxP:

Take a step back and look at the entire workflow. If there are multiple steps, introduce small checkpoints to close these gaps.
Keep revisiting any processes to see if there are any improvements you can make to ensure data security and integrity.
Remember, compliance depends on you, the user. Always use trusted suppliers and validate your workflow thoroughly.

also explains how Cytiva’s Amersham ImageQuant™ 800 GxP biomolecular imagers have a full suite of features.

From biomolecules to celestial bodies:

Uncompromised, high-quality imaging

“If you’re a skilled technician running a western blot, but your system can’t pick up the bands of interest and differentiate these against the background, then you’ll need to repeat the entire experiment. It’s time-consuming and unnecessary,”

Sowmya says. This is especially true if you’re investigating a protein expressed in very small quantities. For effective biomolecule research, labs need imaging systems that can detect low-intensity signals and differentiate them from the background noise.

Traditionally, imagers tend to have a trade-off between resolution and sensitivity. To solve this, Cytiva developed the signal-to-noise optimization watch (SNOW) mode on its ImageQuant™ 800 imagers, which uses signal averaging – a well- known technique used in astrophotography to visualize distant stars. SNOW mode works by capturing multiple images and continuously averaging the signal over time. This process reduces random noise while allowing the signal to remain constant, improving the signal-to-noise ratio.

SNOW mode helps to enhance image quality with reduced noise and better resolution. Find out more about SNOW mode in this application note .

“In terms of performance, the ImageQuant™ 800 hits that sweet spot where you can image difficult-to-see samples and simultaneously have the resolution that you need,” says Sowmya. “But what an industry lab needs more than just an image that gives you good images is a layer of security for its data.”

Data security in the modern, electronic lab

The ImageQuant™ 800 GxP is a new module designed to help users maintain electronic data records in compliance with FDA 21 CFR Part 11 and EU GMP Annex 11 regulations. It includes features like operating system access control, audit logs, and image authenticity checks.

Today’s labs need to consider data security from various angles. If multiple people need access to the imager or its output, they probably need different levels of

access. To address this, the ImageQuant™ 800 GxP lets you assign user groups that grant varying levels access to the required software functions.

The module also tracks and saves a full, time-stamped audit trail of everything that happens in the imager. But beyond imaging steps, it also has features to address larger, fragmented workflows.

“You often start with a sample prep stage somewhere on your lab bench. Then you perform electrophoresis, followed by western blotting, and then you finally reach the imaging step – where you put the blot through your imager before looking

at the image file in your analysis software. That’s when the file leaves the secure environment of the imager,” Sowmya says. “Normally, users record an experiment ID or a sample ID in their electronic notebook at the start of the experiment, but there needs to be a way to seamlessly link up your sample, the imager, and the final analysis steps.”

To link up this fragmented process and ensure consistent electronic reporting, the ImageQuant™ 800 GxP uses a string of “digital handshakes” to connect all stages of the workflow. It uses a mandatory image ID field that helps users trace experiments from the origin of the sample, through imaging, to the final analysis output.

The ImageQuant™ 800 systems promise sensitive, accurate chemiluminescence and fluorescence detection

Each sample gets an assigned ID that you can enter into the imaging system.

This ID connects each sample to its first step, along with everything that has happened previously. Additionally, to reduce the risk of tampering before analysis, images on the ImageQuant™ 800 GxP carry a special security tag within the metadata. The ImageQuant™ TL 10 GxP analysis software reads this tag and alerts you to any suspected tampering – and it won’t open tampered images to better ensure data security.

Future innovations responding to scientists’ needs

Sowmya Balachandran’s top tips for achieving GxP:

Cytiva envisions a world in which human health is transformed through access to life-changing therapies.

Its products and services aim

to support customers in helping advance and accelerate therapeutic development. Its imaging products are used in basic research – including western blotting and gel electrophoresis – to help identify, characterize, and quantify proteins, which is often the first step in any research workflow and serves as a

basis for more advanced experiments that eventually lead to a better understanding of diseases and the effect of drugs on cells.

Another key factor for success is validation. To ensure scientists have confidence, Cytiva provides access to a validation support file on their regulatory support page, which includes full development documen- tation, change control notifications, and external assessment reports.

“The self-checklist inside our validation support file documentation helps users check and validate their workflows

for compliance,” Sowmya explains. “We also went a step further with an external audit that looked at how we developed the ImageQuant™ 800 GxP, what the features are, and checked

all of our system documentation from a GxP perspective. Customers can access this audit report along with the certificate by downloading the validation support file on our website.”

Looking to the future, Sowmya and the team at Cytiva understand that there is always room for improvement. “We are committed to offering the best solution and products to our customers,” concludes Sowmya. “Our product development process is based on customer feedback. This process continued after launch and we always aim to improve our offering, so our customers can always be confident that they have the best-in-class solution with Cytiva.”

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Cytiva and the Drop logo are trademarks of Global Life Sciences IP Holdco LLC or an affiliate. ImageQuant is a trademark of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva.

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Digital handshake to ensure image data integrity: Check image

User

Developer

Admin

Operating system: based login with user groups for controlled access.

Time-stamped event logs: enable easy retrieval for audit trails.

authenticity automatically with ImageQuant™ TL GxP analysis software.

IIDD 123456

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Data traceability: Enter your sample ID and use it to trace your sample.

Electronic record: are embedded with ID an all relevant capture information.

Full documentation and validation support: Sign up with Cytiva for full access to regulatory documentation, like validation

Traceability in Western blot imaging: Considerations for GxP

Densitometry analysis of gels is a key part of quality control (QC) in the pharmaceutical drug release process. In regulated environments, electronic record-keeping can improve both traceability and efficiency – but compliance with regulations like CFR Part 11 and EU GMP Annex 11 is essential.

In this article, we discuss what implications these regulations have on workflows using gel and blot imaging systems to capture, analyze, and document images. We cover what to consider, what to look out for, and how to stay confident that workflows are — and remain — compliant.

Traceability. Accountability. Data integrity.

Around the world, many jurisdictions have their own guidance for regulated environments (sometimes collectively known as GxP), and these regulations extend far beyond the pharmaceutical industry. They help to make sure that only safe, high- quality products reach end users. Though differences exist, all GxP regulations focus on the principles of traceability, accountability, and data integrity.

When you apply these principles to gel and blot imaging — for example, in pharmaceutical QC — you should not only consider the acquisition of the image itself, but the workflow around it, too. Imaging is always part of a broader electrophoresis workflow, so you need to keep all steps in mind when thinking about traceability,

Cytiva

accountability, and data integrity. These steps include sample identification, electrophoresis, image acquisition, image handling and analysis, and data storage.

In regulated environments, many of these workflows are governed by guidelines on electronic records and electronic signatures. In the U.S., it is the FDA’s Title 21 CFR Part 11, Electronic Records; Electronic Signatures - Scope and Application. In the EU, these guidelines can be found in the GMP guidelines under Annex 11: Computerised systems.

Product or process?

There is one key aspect in all these regulations — it is always the processes that need to be compliant, not individual products used in that process. You should never label a product as being certified with CFR or GMP guidelines because the product is part of a process, and it’s the process that needs to be validated.

Instead, we should focus on how well a system supports compliance, because that’s what makes a real difference in the ease of its use. Good support generally includes data traceability, audit trail documentation, and use of user groups. The next sections take a closer look at each of these aspects.

Traceability in imaging

In many gel and blot imaging applications, both the imaging itself and the analysis after have a substantial impact on the conclusions that you can draw. For example, in pharmaceutical QC labs, gel images of drug batches are a key factor in determining drug purity levels. In turn, the drug purity levels affect the main decision

— can this batch be released or not?

As imaging systems and image analysis software evolve, we have increasingly more ways to maximize information from a subject. In regulated environments, the goal is to maximize data quality without compromising on consistency or traceability

in any way. The main way labs can achieve this is by making sure that image files contain a full history of how the image was taken. That can include:

Date and time of image capture
The user taking the image
Details of instrument taking the image
Imaging mode and capture settings (exposure time, binning, etc.)
Name of method used to take the image

This information is contained in metadata that the system generates together with the original image in the TIF tags. You can find the tags by viewing the image

properties, enabling users to easily trace the image back to its origin and get valuable details on its capture.

Keeping track throughout the workflow

Image metadata is a key contributor to traceability, but it’s certainly not the only one. Samples and their resulting data need to be traceable throughout the entire workflow. With unique sample IDs, scientists can track a single sample across different steps of a seemingly scattered workflow.

Another important aspect is data security when moving from one step to the other. For example, imaging and image analysis often happen at different times and in different locations, leaving a gap between the secure environment of the imager and the secure environment for analysis. What happens to the image in between? How can you tell if it’s been tampered with or not?

One way to be sure is by using analysis software that checks for image authenticity. The specialized software works by recognizing a security tag embedded in the image. If the image was altered between imaging and analysis, the software takes note and treats the image differently than it would handle an authentic image.

Audit trails

The result of the comprehensive, secure tracking of activities from the time

the imager is turned on is that it should be easy to generate a reliable audit trail. Audit trails are a key requirement in many GxP regulations. For example,

CFR part 11 states:

“Use of secure, computer-generated, time-stamped audit trails to independently record the date and time of operator entries and actions that create, modify, or delete electronic records. Record changes shall not obscure previously recorded information.” —21 CFR 11.10e

With appropriate imaging equipment and analysis software, this type of documentation is fast and easy to generate when requested. Remember that documentation showing traceability does not begin at imaging — sample preparation and electrophoresis are both critical to the final image, and details of these parameters should be recorded. It also helps to assess how well you can integrate

the imaging audit trail into the documentation for the rest of the workflow.

User access

The way in which users access images, protocols, and documentation is another area where GxP labs are fundamentally different from most other labs. For example, regulations can control how users can get access to a system, as well as which users can access different features. Controlling access helps to minimize errors, improve transparency, and reduce opportunities for malpractice.

In a compliant workflow, system access is unique for each user and uses two components of authentication (e.g., username and password) — although a single component suffices for additional logins within a continuous period of controlled system access.

To reduce ambiguity further, the system should avoid conflicts in user access. For example, the system wouldn’t let a user log into the computer’s operating system when a different user is logged into the software. To do this, scientists can align the imaging software’s login protocols with the Windows™ operating system, or take other steps to prevent these conflicts from happening.

Sometimes, users might want to leave the imager during image acquisition. A helpful

feature that supports this without risking security is letting users lock the imager without interrupting the exposure.

GxP regulations also provide guidance regarding different types of access for different users. Separate access rights — different rights for users, method developers, and administrators — helps to ensure that only personnel with the right qualifications can carry out certain operations. For example, basic users often don’t need to change protocols, so it makes sense to restrict their ability to create new or amend existing methods.

Summary

The role of electronic processing and documentation is rapidly expanding, and GxP-compliant labs can benefit greatly from digitized systems. Scientists working in a regulated environment have specific requirements when choosing new equipment, and those requirements are different from most other labs. The ease with which a system maintains and demonstrates compliance is key.

Data traceability, audit trail documentation, and defined user access rights are the critical elements to get right. Combined, they can create the confidence that innovation and compliance can go hand in hand.

At Cytiva, we are passionate about supporting scientists regulated environments with their needs and goals. To this end, we offer a gel and blot imager along with image analysis software with a host of features designed to help users demonstrate full compliance with GxP regulations.

Our Amersham ImageQuantTM 800 GxP biomolecular imager and ImageQuant TL GxP analysis software incorporate the core principles of traceability, accountability, and data integrity — helping scientists and regulators deliver safe and effective drugs.

Title

A C

D E F

G H I

Science meets art: Cytiva’s ImageQuantTM 800 reveals hidden detail in these images across the EM spectrum from UV to infrared. (A) Daisy (Bellis perennis) (B) Mineral sample (C) A cowslip (Primula veris) (D) A dandelion (Taraxacum officinale) (E) A shrimp (F) A mineral sample (G) A wood anemone (Anemone nemorosa) (H) A siberian squill (Scilla siberica) (I) A yellow wood anemone (Anemone ranunculoides).

Understanding 21 CFR Part 11 and its application in analytical labs

Validation, compliance, and international regulations expert, Sion Wyn, assisted the FDA with its re-examination of 21 CFR Part 11 and was part of the core team that produced the FDA Guidance on 21 CFR Part 11 Scope and Application .

“To achieve efficient compliance, it is essential for regulated companies to understand and define their processes, recognize the applicable predicate rules, and identify their Part 11 records and signatures. Then they must identify and apply the appropriate Part 11 controls based on a justified and documented risk assessment.

I am extremely proud to have been a core team member and editor for the FDA Guidance for Industry Part 11, Electronic Records; Electronic Signatures - Scope and Application, which aimed to avoid unnecessary controls and costs, and to encourage innovation and technological advances in this area.”

Sion Wyn, Director of Conformity Ltd.

With the increasing use of computers to generate, store, and manage experimental data, standardized regulations are needed to address potential security concerns and protect data.

The 21 CFR Part 11 regulations issued by the U.S. Food & Drug Administration (FDA) aim to establish criteria for the acceptance of electronic records and signatures as equivalent to paper records. An ‘electronic record’ is defined in Title 21 - Food and Drugs, Chapter 11.3 (6) as “any combination of text, graphics, data, audio, pictorial, or other information representation in digital form that is created, modified, maintained, archived, retrieved, or distributed by a computer system.”

If your company is FDA regulated and is uploading or storing GxP records on a computer, it is almost certain that 21 CFR Part 11 compliance regulations

apply. Users must understand and interpret these guidelines to the best of their knowledge and establish a thorough analysis of their relevant processes and validate to ensure compliance.

“Good practice” (GxP) and 21 CFR Part 11 regulations can broadly be interpreted within three categories: accountability, traceability, and data integrity. It is important to maintain these facets from the very start of the workflow, which can be challenging due to the dispersed nature of gel electrophoresis and western blotting processes.

While reviewing your workflow for 21 CFR Part 11 compliance, it is important to ensure chain of data custody, evaluate the risk (probability and severity) of failure,

and evaluate possible control measures that can be introduced to minimize the risk for each defined step or sequence.

Start by defining applicable workflows that describe the sequence of activities performed in your lab. In an analytical lab, the experimental workflow may consist of multiple sample-handling steps, experimental methods, and/or use of instruments and reagents. Compliance is therefore not limited to just buying and

installing software or products that support 21 CFR Part 11 compliance. The entire workflow and its instrumentation need to be verified.

A typical drug purity testing workflow consists of multiple steps:

Fig 1 . After sample preparation and gel electrophoresis, the gels are stained with dye

(e.g., Coomassie™ Blue), imaged on an imager (e.g., ImageQuant™ 800), and finally analyzed with software (e.g., ImageQuant™ TL). It important in a regulated environment to maintain data traceability through these steps to be able to ensure confidence in results generated.

10 key considerations when choosing equipment for your regulated QC lab

Accountability of the electronic record
It is important that you can identify who has contributed what, and when, and that this information can be easily viewed when needed. One way equipment manufacturers achieve this is by embedding the relevant information within the electronic record itself.
In imaging systems, the generated .TIF file contains multiple embedded ‘tags’ that can provide information including when the image was taken and where.
In Cytiva’s Amersham ImageQuant™ 800 GxP biomolecular imagers, the captured .TIF image files are the primary electronic record and each image file contains all the relevant capture information, including username, date and time, capture settings, version of software and hardware, etc. These can also be easily viewed within the software during an audit process.
Authentication and identification
Access into any system must be controlled by a unique username and password for all users. Part 11 discusses a continuous period of controlled system access by users.
It is generally good practice to have laboratory equipment (like CCD imagers) that are controlled by an external computer and use Windows domain-based logins.
In systems such as the ImageQuant™ 800, authentication is secured by ensuring that an individual uses their Windows® credentials to log into the ImageQuant™ 800 GxP control software. This also allows the control software to directly follow any user login rules set by the organization, such as a password expiration policy, and eliminates the need for any separate rules within the control software of the equipment.
Restricting system access with user groups

User groups help to define the role of personnel using the equipment and enable access to certain system capabilities based on this role. This ensures only relevant personnel have access to certain functions.

In the ImageQuant™ 800 GxP imager, the administrator controls the creation of accounts,

a developer makes standard operating procedure (SOP) methods, which are then performed by a user. It is possible for one user to be assigned multiple roles, especially

in smaller companies where the lab manager could act as both administrator and developer. In this instance, any potential risk of conflict of interest should be considered and managed accordingly.

4 . Event logs for audit trails

The event log onboard an instrument allows for traceability of all relevant events on the imagers when in use. This history of events allows heads of department and supervisors to perform checks on a regular basis and external auditors can use this for formal audits. Event logs must be readable, tamper-proof, and available for export for filing and maintaining records.

5. Data traceability

It can be challenging to trace back to the very first step of an electrophoresis or western blotting workflow as these processes involve multiple smaller experiments and steps.

However, you can create a chain of custody by giving the sample an ID at the start of the workflow, recorded in an electronic notebook, that follows the sample straight through image generation and data analysis.

The ImageQuant™ 800 GxP contains a mandatory sample or image ID field so this information can then be embedded in the .TIF tag. It can be viewed under image properties

anytime and always stays within the image’s metadata. This creates sample data traceability and acts as a virtual handshake between experimental steps.

6. Data integrity

Data integrity is crucial to any experimental result, especially when the final exported data is an image file. There are multiple software solutions that can edit or tamper with an image file and it is therefore important to always ensure that images are not tampered with prior to analysis. The ImageQuant™ 800 and ImageQuant™ TL GxP analysis software performs an integrity check at the start of analysis to ensure this.

7 . Digital signatures where applicable

Electronic signatures can be used in place of paper and pen signatures to enable electronic record approvals. Electronic signatures must be unique to one individual, must not be reused, and must contain the date and time of the signature. Many systems allow for these electronic signatures where required – usually where approvals or data review is needed.

Some systems only generate images and do not need electronic signatures. However, image analysis software requires electronic signatures as analysis is performed, reviewed, and finally approved by different people in the organization.

8. Equipment qualification services

It is important to ensure that analytical lab equipment is tested at the time of installation and every few years (depending on your company policy). Instrument manufacturers develop and approve testing protocols and certified service engineers then perform these in alignment with GAMP5, ICH Q1-10, and ASTM E2500 standards. Qualification at the time of installation is called IQ/OQ (installation qualification/operation qualification) and repeat testing every few years is called RQ (requalification). Companies like Cytiva provide qualification services to support your equipment throughout its lifecycle.

9 . Documentation support

Since the final responsibility for the 21 CFR Part 11 compliance claim rests with you, the user, supporting documentation provided by some instrument manufacturers can prove valuable and help you support 21 CFR Part 11 compliance for your processes.

These documentations could be user guides or operating instructions specifically for GxP environments, assessment reports or validation support documents. Cytiva provides

the Validation Support File to all users of the ImageQuant™ 800 GxP which contains information on development, a self-assessment checklist and an external 21 CFR Part 11 Audit assessment report.

10 . Knowledge and support

Finally, it is important to make sure that the manufacturer of your chosen instrument has experience developing and maintaining equipment for regulated environments and a strong knowledge of the different aspects of GxP. This ensures confidence when selecting products and provides reliable support throughout the life of your equipment.

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Ensuring GxP compliance with the Amersham ImageQuant™ 800 GxP biomolecular imager

Watch webinar on demand

As more processes go digital, laboratories are having to move away from time-consuming procedures and the use of paper records to ensure better data integrity and traceability.

Good practice (GxP) regulations require that drug manufacturers maintain records of every step of the workflow and include guidance on how to ensure their equipment is validated and compliant with the requirements of FDA 21 CFR part 11 and EU GMP Annex 11.

Watch this webinar to learn more about:

GxP regulations for safe electronic record keeping
Protecting raw data, checking file authenticity and tracing image history
The features of the Amersham ImageQuant™ Biomolecular Imager that supports electronic data management and data compliance

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Cytiva and the Drop logo are trademarks of Global Life Sciences IP Holdings Corporation or an affiliate.

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