Whether characterizing particle size or structure, or investigating any possible contamination, particle analysis plays a critical part in bio/pharmaceutical development and manufacture. “Particle analysis is a critical tool to define, help manage, and deliver product quality,” confirms Michael Lindsay, SGS Global scientific director.

“Desired product performance greatly influences how the API needs to perform with regards to solubility, rate of solubility, and permeability to access the target site or be available for systemic circulation,” Lindsay continues. “Typically, the API is blended with other non-active ingredients, each with their own contributing property attributes, to help deliver the API in the desired media to a patient as a solid, semi solid, liquid or gas/powder.”

Essentially, achieving a sufficiently homogenous mixture and product is a key manufacturing goal, but there are multiple characteristics that can impact this, such as physical aspects (size, shape, density), particle charge, smoothness, and the ability to blend and flow, Lindsay notes. “These features can be critical quality attributes in process steps from: initial synthesis through crystallization, if needed; processing and blending in production into the final package to contribute final product performance,” he says. “Collectively, these factors influence which particle analysis methods are used to evaluate material behavior to determine criticality; and, with defined specifications become necessary to ensure consistent product quality and performance.”

During manufacture, drug products come into contact with other surfaces and can be exposed to the air, which could lead to potential contamination from particulates, Lindsay asserts. “Therefore, particle analysis also serves as an investigative tool to ensure there are no unacceptable contaminants,” he says. “Where contaminants are present, particle analysis helps identify root causes for such contaminants, so that actions are taken to eliminate them to further ensure product safety.”

There are numerous techniques that are currently available for particle analysis, which can be categorized around both chemical and physical properties, Lindsay explains. Additionally, it is possible to use single or multiple methods to reveal the expected materials performance so that an acceptable product can be yielded, he adds.

 “Material solubility is a significant contributor to product performance where particle size reduction corelates with increased surface area to influence rate of dissolution. Thus, multiple methods are used to measure particle size,” Lindsay says. “A desirable decrease in particle size can facilitate higher dissolution with increased surface area and can also increase the rate of degradation and impact product stability too. Therefore, particle size analysis is one of the most important techniques.”

A relatively easy method that requires minimal expertise and no sample preparation is sieving, which includes air jet sieving, specifies Lindsay. However, with this method between 10 g and 100 g of material is consumed, there is a lower particle size limit (>20–50 µm), the range of sieves reduces data points, and it is only possible to be used with dry materials, he states.

Through the use of light microscopy with high resolution-image analysis, it is possible to fully visualize and inspect particles, Lindsay confirms. This method provides such advantages as a 1–1000 µm dynamic range, visualization of solubility (if it occurs), capability of confirming aggregation and particle fragmentation, and the ability of confirming crystallinity, degree of crystallization, crystal quality, and shape. Considerations to take into account when using this approach are solvent evaporation; multiple fields of view; and non-random particle orientation if particle size or shape is also being measured.

Laser diffraction only requires a small amount of sample (>10 mg to a few grams), can be used with wet or dry particles, and offers a dynamic size range of 0.01–2500 µm (dependent on instrument, technique, and configuration of instrument), Lindsay continues. For wet analysis, however, the solubility of the material should be considered, he adds. Additionally, expertise for method development is required, and the particle shape needs to be contemplated when the technique assumes a sphere.

A small quantity of sample (±1 mg or less as a minimum) is also required when using dynamic light scattering (DLS) as a technique for particle size analysis. The dynamic range offered is between 0.3 nm–10 µm and the estimated average molecular weight range catered for is 1 x 103 to 1 x 107 Daltons (both dependent on instrument, technique, and configuration of instrument), Lindsay states. Similar considerations are required for DLS as with laser diffraction: solubility, expertise, and particle shape.

Dynamic image analysis or Micro-Flow Imaging (ProteinSimple, Calif., USA) is another particle size technique that only requires a small quantity of sample (<1 mL). This approach offers a dynamic range of 1–300 µm (dependent on instrument, technique, and configuration of instrument), provides the ability to see particles, allows visualization and measurement of particle shape and morphology, tests most of the sample, can be used with high viscosity samples (up to 20 centipoise), and can enable visualization of contaminant particles, Lindsay asserts. Solubility and expertise are considerations to take into account with this method, along with the need to view sufficient images for confirmation of particle distribution because particles are visualized in a random orientation.

“A consideration for all methods discussed is that the sample tested must be representative of the bulk; particle size is measured through a variety of techniques, where in development orthogonal approaches may be taken to evaluate which provides the more meaningful data to control quality,” Lindsay stresses.

 “Other techniques can be employed to visualize particles, isolated or not, to better describe the particle and aid in identification of potential contaminants,” states Lindsay.

When evaluating surface area of particles, it is possible to use the Nitrogen Brunauer-Emmett-Teller (BET) method. “Surface area most influences solubility of a material in solution and can be additionally measured as an orthogonal technique to particle size,” Lindsay says. “As particle size decreases surface area increases per unit mass and reported as m2/g to positively increase potential dissolution rate. Thus, particle size/surface area is used to help control dissolution rate in some products.”

Measuring the surface area of particles becomes particularly important for chemically unstable compounds, Lindsay explains. For these compounds, if there is an increased surface area there will be an increased degradation rate, which influences the formulation protection or coating that has been implemented to prevent degradation.

Surface charge method can be used to evaluate the Zeta potential—the charge on a particle at the shear plane. This property is important for the understanding and prediction of how particles will interact in and successfully stay in suspensions with other particles, Lindsay confirms.

Flowability of particles, which is critical for powder blends, can be measured with a variety of methods, such as funnel, angle of repose, powder viscometry/rheology, and so on. “The ability to flow evenly is a requisite for gravity assisted processes, where flow is uneven or there are vibrations differences in flow, density, particle size can lead to size or material separation to de-mix a once uniform blend,” Lindsay says.

Pycnometry/a densitometer can be used to measure the material density at a material level and to measure that property at a bulk scale, it is possible to use tap and bulk density testing. The latter method can also provide details that will allow for an understanding of how particles will settle together, Lindsay notes.

 All products to be administered via injection need to undergo a visual inspection to check for particulates as per regulatory guidance, such as the United States Pharmacopeia Chapter <790> and European Pharmacopoeia Chapter 2.9.20 (1,2). The visual checks involve an inspection of the sample against a black and against a white background, with the naked eye and without magnification, under standardized light conditions, Lindsay states.

Light microscopy can be used to visualize the particles and their surface properties to a size of 1 µm. Additionally, this technique allows for particle geometry to be evaluated and measured, and can provide confirmation of crystal properties, Lindsay reveals. A simple count of particles in liquid samples, at set sizes between 10 µm and 25 µm, can be done using light obscuration, he adds.

Microscopy coupled with Fourier-transform infrared or Raman spectroscopy may be used to identify particles greater than 10–20 µm, although particles must be cleaned of other materials to improve spectral quality, Lindsay stresses. Scanning electron microscopy and energy dispersive X-ray analysis are useful for the visualization of the material and for elemental identification, particularly for metal/alloy contaminants.

“Particle analysis techniques have advanced greatly from the simple sieve to the use of laser and high-speed image and spectroscopic systems to gather more meaningful sample data on the particle to better describe the particle shape, size, density, and identity faster,” says Lindsay. “Also, providing the sample is representative, results are obtained with much less material thus enabling accurate measurements on very expensive materials at lower cost.”

Thanks to advancements in optical and spectroscopic techniques, it is now possible to capture images of the particle flow in real-time, allowing for particle identification, Lindsay reveals. Additionally, smaller particles (>0.3 nm) can be optically detected and measured using light scattering techniques, he adds.

“Data processors, detectors, and processing speed have contributed significantly to the advancement of new instrumentation that are both smaller and faster than previous instruments,” Lindsay says. “Bench instrument components are now being built into flowing or blending processes to also enable real-time in-situ non-destructive analysis and include final in vial measurements so that no additional material is consumed. Spectroscopic techniques, with complex libraries and processing for spectral analysis, enable real-time monitoring of blends to confirm material concentration and uniformity.”

In the future, continued advancement in analytical measurement abilities for all aspects of particle analysis to the limits of what physics will allow is expected, Lindsay emphasizes. Further miniaturization of instruments to enable measurements of lower volumes and greater speed in flowing systems and an increased use of process analytical technology (PAT) solutions that separate offline analysis will transpire, he states.

“In time industry should expect to see more analytical instrument techniques be part of the PAT solution to reduce some laboratory testing outside of the particle analysis areas too,” Lindsay adds. “The regulations already support alternate techniques to better manage risk and assure quality. Data analytics with smarter artificial intelligence will help identify and correct processes, within defined and approved design and quality-by-design spaces, seamlessly to enable more effective production.”

 <790>, “Visible Particulates in Injections,” 

 Chapter 2.9.20, “Particulate Contamination: Visible Particles” (Strasbourg, France, 2008).

Felicity Thomas is the European editor for Pharmaceutical Technology Group.

Articles

Features

Particle analysis provides assurances of the quality and performance of the final dosage form in pharmaceutical development.

Without a Particle of Doubt

Biopharmaceutical analysis is one area in biopharma drug development that requires improvement, namely to manual processes that take up significant amounts of time and labor. Sample preparation remains a major bottleneck point to productivity because of the number of manual steps involved and, in some cases, the outdated technology used to ready samples for analysis.

Glycosylation monitoring is one aspect of biopharma analysis that falls under the strain of laborious manual processes. However, advances made in technology and approaches to automate the workflows for glycosylation monitoring offer quicker sample preparation and can cut down on overall time needed for sample analysis.

Because many recombinant biotherapeutics, such as monoclonal antibodies (mAbs), are glycoproteins, monitoring glycosylation activity is necessary for biopharma drug development. Bioprocessing requires precise control and monitoring of multiple parameters, including cell line stability, product yield, protein folding, and post-translational modifications (PTMs), specifies Ning Zhang, PhD, senior product marketing manager, Chemistry Technology Center, Waters Corporation.

Among the parameters of interest, the host cell’s biosynthesis of glycans is routinely monitored as a critical quality attribute (CQA) because glycan composition and structure are crucial for the biological or clinical activity of the drug. “Slight changes in manufacturing conditions can alter the glycosylation patterns of recombinant proteins and, consequently the biological activity, safety, stability, efficacy, and immunogenicity of the end drug product,” explains Zhang. The manufacturing of therapeutic glycoproteins, therefore, requires careful monitoring and characterization of their glycosylation profile to achieve consistent glycan composition and meet desired quality, clinical safety, and effectiveness targets.

“Protein glycosylation plays an important role in bioactivity, stability, biological half-life, immunogenicity, and pharmacokinetics of a biopharmaceutical,” confirms Radoslaw Kozak, PhD, head of Glycoprofiling at Ludger. Kozak explains that glycosylation is a PTM and, unlike transcription, is a non-template-driven enzymatic modification process. Glycosylation of proteins can therefore change with alterations to production conditions.

Glycosylation could be considered the “canary in the coalmine” for monitoring the consistency and reliability of a manufacturing process, adds Amy Claydon, PhD, Scientific Account Manager, Genedata. She explains that even slight changes to the bioreactor conditions during protein expression can lead to measurable differences in the glycosylation profile.

“The presence, or absence, of glycosylation modulates antibody activity by enhancing either antagonist (blocking, inhibiting) or agonist (activation) functions. Subsequently, glycans may impact the safety, efficacy, structure, and circulating half-life of biotherapeutics; for example, a mAb with no glycosylation will not fold correctly and so will be ineffective as a treatment. This effect on the immunogenicity of a therapeutic antibody increases the risks for patients,” says Claydon.

Claydon further explains that pharmacokinetics, especially clearance and circulating half-life, are also affected by N-glycosylation. For instance, higher levels of sialic acid-containing glycans can increase the circulating half-life because these glycans become more resistant to removal by hepatic asialoglycoprotein receptors.

Dealing with processes in sample preparation for glycosylation monitoring that currently depend on manual labor tends to be a bottleneck point in productivity. To start with, there are several analytical approaches used for the analysis and characterization of protein N-glycans during process development, notes Zhang.

One common approach involves the enzymatic release of N-glycan chains from the protein of interest and their derivatization with a fluorescent label. “A manual procedure, this conventional deglycosylation method requires the incubation of a glycoprotein sample for about one hour, while many researchers generally employ an overnight (16 hours) incubation. This is followed by a lengthy two-to-four-hour labeling step based on reductive amination reactions,” explains Zhang.

The analysis of released N-glycans is challenging because of the number of steps that are involved in preparing the glycoprotein samples and processing the released glycans to prepare them for analysis, adds Richard Gardner, PhD, lead scientist, Ludger. The release of peptide-N-glycosidase F (PNGase F) and the fluorescent labeling of N-glycans, followed by a cleanup, are necessary steps for N-glycans to be analyzed. Gardner points out that these steps are challenging and time consuming when performed manually, especially when the numbers of samples increase beyond a certain amount. “This makes it more difficult for the user to accurately pipette low volume solutions and to efficiently track which samples have been processed,” he states.

Each stage needed for optimizing sample preparation for N-glycan analysis requires on-going evaluation to determine effectiveness, says Claydon. This can subsequently lead to lengthy assay development times if rapid data analysis techniques are not available. “Of course, there are also numerous methods to implement automation (robotic liquid handling, for example) in sample preparation itself, but these often can highlight process inefficiencies further along the process too,” Claydon says.

Screening necessarily large numbers of samples can be time-consuming and monotonous, says Zhang. Furthermore, methods for glycan sample preparation and analysis must be robust and reproducible to ensure accuracy and consistency, she adds.

Automating different aspects of a workflow is one method to alleviate the bottlenecks caused by processes dependent on manual labor. Automated data analysis, for example, can improve many areas of glycosylation monitoring workflows, says Claydon. “When automated data analysis is employed, it can quickly facilitate understanding the effects of different sample preparation optimization steps—the impact of altering digestion conditions and so on. This, in turn, can lead to more rapid improvements to the digestion and labeling protocols during the method development process and an overall increase in assay throughput,” she states.

Bottlenecks often go “hand-in-hand”, Claydon also notes, and solving one bottleneck often leads to another in a different part of the process. “This is why we like to look at data solutions as part of facilitating the whole analytical workflow. In this example, we can easily predict that making sample preparation faster in some way (e.g., automated robotics) can create a lot more data that then needs to be analyzed. So, it is not a simple case of adding automation in just one part of the workflow that ultimately makes the challenges easier.”

Another instance of laborious manual work centers on the fact that many of these workflows have multiple pipetting steps in a range of volumes, which takes time. Automation can save time and allow for more reproducible liquid handling, says Zhang. In addition, automation allows multiple samples to be prepared in parallel, thus improving the throughput.

“Standardized sample preparation is essential to minimize user variations and pipetting errors and to ensure that glycan data is accurate and reproducible. Minimal user intervention—or none at all—allows users to stay away from the bench and work on documentation or setting up an instrument while preparing the samples,” Zhang emphasizes.

Gardner concurs that automation would alleviate this bottleneck by allowing a higher throughput of samples where user intervention is minimized. “Automation would improve the repeatability, reproducibility, and robustness of N-glycan processing by removing the user pipetting steps, thereby decreasing possible human error when transferring the small amounts of liquids that are used in these methods. One outcome would be a harmonized method that many users can operate that removes potential differences in the way users work and interpret SOPs [standard operating procedures],” he notes.

Some technological advances have been helpful in easing the incorporation of automation in the glycosylation monitoring workflow. For instance, specialty sample preparation kits (GlycoWorks RapiFluor–MS, Waters) available commercially and an associated workflow can make fast work of de-glycosylation and labeling. This reduces sample preparation time from a day to less than one hour while enhancing the sensitivity for both fluorescent and mass spectrometry (MS) detections, notes Zhang. “Reagents and reaction conditions that come with these kits have been optimized to enable a fast and complete de-N-glycosylation of glycoproteins and subsequently the rapid and efficient labeling of released N-glycans. Following a HILIC [hydrophilic interaction liquid chromatography]–SPE [solid phase extraction] sample clean-up step, the samples are then ready for analysis by LC–MS [liquid chromatography–mass spectrometry],” Zhang says.

The main technological advance in the field of automation, notes Gardner, is the introduction of robotic liquid handling platforms. Gardner explains that the incorporation of robotic liquid handlers such as Hamilton’s Microlab STAR into glycosylation analysis workflows has allowed many of the processes to be automated, from the release of N-glycans using PNGase F to fluorescent labeling of N-glycans all the way through to preparation of samples for LC–MS analysis.

“Many larger robotic platforms including Hamilton’s Microlab STAR not only incorporate liquid handling and vacuum manifolds but also incorporate plate sealers, heater shakers, and the ability to move plates off deck for centrifuge steps. This ability to incorporate different lab equipment into a robotic workflow enables the move towards full automation of glycosylation analysis. Laboratories require an automated system that is simple to operate, scalable, and repeatable,” Gardner states. To facilitate the move to higher throughout and automated analysis, a range of products have been introduced onto the market that are compatible and adaptable to many robotic platforms, including a 96-sample recombinant PNGase F kit, a 96-well protein binding membrane cleanup plate, a 96-sample procainamide glycan labeling kit, and a procainamide cleanup plate (Ludger).

Meanwhile, Claydon says that automation is best thought of as an end-to-end process, and we must therefore consider how the introduction of automation in one area may move bottlenecks to another part of the overall workflow. “If one considers that an automated data analysis platform actually facilitates both upstream and downstream areas of the entire assay, then it can be a tool to help fully understand where bottlenecks may have moved to, and, in doing so, then accelerate optimization to reduce the time spent in other areas, if they do not significantly affect the end result. For example, long chromatography runs can be avoided if the challenges introduced by co-eluting peaks can be later simplified using software that can display the data two-dimensionally, and deconvolute them,” she states.

“The number one request we get is for help with automating the liquid handling steps, essentially the pipetting of sample preparation protocols,” adds Zhang. “Liquid handling devices now come in several forms including robots (Andrew+ Pipetting Robot, Waters) that automate many sample preparation steps including pipetting which, as everyone knows, is tedious.”

In addition, according to Zhang, robotic pipetting systems can prepare up to 48 samples in under 2.5 hours. Greater automation is achieved by adding connected devices for heating/cooling and a vacuum for sample clean up with SPE.

There are many ways that samples can be prepared for glycosylation analysis, according to Kozak, who explains that some samples may need filtering and/or buffer exchange while other samples may need an enrichment step to extract the glycoprotein to be analyzed. Technologies using 96-well plates that incorporate filters or protein capture technology have allowed this type of processing to happen on automated platforms, Kozak states.

Employing a fully automated liquid handling robot is one innovative approach for automating and streamlining routine sample preparation steps, Zhang adds. This improves both reproducibility and sample throughput. Another innovative approach is by means of a guided pipetting system (Andrew Alliance Pipette+, Waters) that features Bluetooth-connected “smart” pipettes connected to a personal computer or tablet via software for designing, sharing, and executing protocols with audit trails, she also notes.

Meanwhile, the ability to routinely monitor many glycopeptides independently using automated LC–MS analysis offers the ability to trend and compare various aspects of glycosylation, says Claydon. “If one were to observe a change in the sample-to-sample response for specific glycopeptides, for example, the signal for one glycopeptide remains stable but that of a different glycopeptide (or a combination of glycopeptides) gives a varying response, then site-specific information can be derived,” she states.

Feliza Mirasol is the science editor for 

Sample preparation tends to be manually labor intensive, but automating this step helps streamline the glycosylation monitoring workflow.

Automation Helps Streamline Glycosylation Monitoring Workflow

Enzymes—the biological catalysts that accelerate chemical reactions inside cells—play crucial roles in every living organism. The catalytic activity of these protein molecules is widely recognized, speeding up reactions with high specificity and efficiency under mild conditions.

Over recent decades, the use of biocatalysts has rapidly expanded across multiple industrial processes. These enzymes are divided into six classes based on the types of reactions they catalyze—hydrolases, oxidoreductases, transferases, lyases, isomerases, and ligases/synthetases. They are used in the form of cell lysates, whole cells, or purified enzymes, prepared either from their native cells or using recombinant technology in alternate host cells.

Enzyme-based catalysis can offer significant advantages over the use of conventional chemical catalysts. Biocatalysis not only helps speed up manufacturing processes but can also improve quality, reduce waste, and optimize product yield, ensuring greater cost-efficiency and increased profitability.

In the biopharmaceutical and pharmaceutical industries, enzymes are fast becoming the catalyst of choice for the development of new drugs, vaccines, and diagnostic kits. Their potential advantages include exceptional specificity towards recognized substrates, simplified drug synthesis routes, and providing an opportunity for a more sustainable and consumer-friendly approach by replacing harsh chemicals typically used in the vaccine production process.

Despite the clear benefits of biocatalysis, the ability to identify, obtain, test, and optimize enzymes with suitable characteristics for specific applications remains challenging. Advances in screening technologies and bioinformatics are fueling the rapid discovery of new enzyme variants. The application of protein engineering supported by computer modeling is also expanding opportunities to modify the structure of an enzyme to try to improve its properties.

Every candidate enzyme will work best under a specific set of conditions, which need to be carefully defined by R&D laboratories. But traditional assays used to assess enzymatic activity have significant limitations. With increased demand for the use of enzymes within bio/pharmaceutical production, there is a need for accurate and streamlined enzyme activity and enzyme kinetics analysis.

Enzyme assays are laboratory methods for measuring enzymatic activity and are vital for the study of enzyme kinetics and enzyme inhibition. Activity is monitored by measuring the decrease of substrates, or increase in products, over a defined amount of time. Usually, the assay is carried out by determining the enzyme activity with, and without, activation by an added coenzyme.

Enzymes work by binding to their specific substrates and forming an enzyme-substrate complex. Within the specific reaction, the substrate is converted into the product(s) which is/are then released. Enzymatic activity measures the rate of an enzyme reaction and is generally defined by the amount of substrate converted (or product formed) in a specific amount of time.

Enzyme kinetics describe the speed at which an enzyme-catalyzed chemical reaction proceeds. The speed of the reaction depends on the amount of enzyme used as well as the amount of substrate. While speed increases proportionally to the enzyme concentration, adding more substrate will initially increase speed linearly; eventually, however, the reaction reaches a maximum speed.

Most enzyme assays are traditionally based on spectroscopic techniques, with absorbance and fluorescence the most used types. Due to its low cost and relative simplicity, the spectrophotometric assay remains the most widely used method (1). This classic enzyme test involves following the course of a reaction by measuring the changes in the intensity of light absorbed or scattered by the solution.

Enzyme assays based on photometry, fluorometry, 96-, 384-, or even 1536-well microplate formats offer a high-throughput alternative to traditional spectrophotometers. However, these methods have limited incubation temperature, temperature stability, and precision due to edging effect, limiting their range of applications.

Measuring enzyme activity is a precise job and can be influenced by many variables, such as temperature, pH, buffer type, or ionic strength (

). Ensuring all these variables are fixed is critical for achieving reproducible results in enzyme assays. For instance, the accuracy of the results is highly dependent on temperature stability. Just a one-degree change in temperature can lead to a 4–8% variation in enzyme activity. Adding the sample, buffer, and substrate in a specific sequence and time interval is also critical for achieving accurate and reliable results.

The major disadvantage of traditional spectroscopic techniques is that all steps involved are manual and can lead to inconsistent results. Based on the enzyme type, the process could take 30 to 60 minutes per enzyme from sample start to data collection, which severely limits throughput. These methods are only suitable when analyzing a few samples or carrying out routine tasks.

While the microplate format approach offers a convenient alternative for high-throughput analysis, this method suffers from temperature stabilization, absorption correction, and edge effects. As the absorbance is measured vertically on microplates through the well, several factors affect the liquid pathlength, and, therefore, the absorbance. As a result, in photometric microplate measurements, a pathlength correction is required for calculating the enzyme assay, and the primary cause of the “edge effect” phenomenon, which is commonly associated with 96-well plates, is attributed to an increased evaporation rate in the circumferential wells compared to those in central locations.

For some enzyme assays, it is necessary to quench or stop the reaction at a specific time to prevent further generation of the product. For example, samples may be collected every five minutes for a predetermined amount of time with the product then measured using high-performance liquid chromatography (HPLC), with each analysis taking 30 minutes to complete.

Developing a reliable analytical method for an enzyme assay involves many steps and often requires extensive work (

). Overall method development is laborious and time-consuming, taking four to six weeks even for highly-trained personnel. The process starts with identifying the key method variables, which requires the testing of many sets of samples to determine the optimal experimental conditions. There is a clear need for a new method utilizing automated solutions that can overcome the key challenges associated with traditional approaches for enzyme analysis.

Streamlining enzyme analyses with discrete analyzer technology

The introduction of innovative and dedicated discrete analyzers offers opportunities to streamline and simplify enzyme analyses. These dedicated discrete analyzers (enzyme analyzers) can efficiently manage the simultaneous analysis of many different enzymes and measuring conditions in a single instrument that offers consistent and reliable performance. All the necessary enzyme analysis steps are automated, which helps to control the variables and makes the overall method development easy and repeatable.

Fully automated enzyme analyzers offer several useful advantages over more traditional methods for enzyme assays. First and foremost, these instruments provide the opportunity to fully automate the critical steps involved in reliable enzyme analysis. For consistent results, an assay should be carried out in well-defined conditions that can be duplicated in other laboratories. Precise temperature control within ±0.3 oC eliminates the risk of sample overheating along with accurate incubation time. Superior temperature control, along with the lack of edge effects, can assure confidence in results. Precise liquid handling (sample, buffer, and substrate addition and mixing) can also improve reproducibility between experiments without compromising on throughput (2).

The technology also enables increased productivity. Within the instrument, samples are simultaneously prepared and queued, delivering one set of results after another. This makes it possible to evaluate many different enzymes, substrates, and methods more quickly and easily (2).

A fully-automated workflow also enables simpler, faster overall method development. Once the testing workflow is set up, the instrument can be left unattended, freeing up time for laboratory staff to carry out other important tasks. Thanks to dedicated software that accompanies enzyme analyzers, workflows are simple and there is practically no changeover time from one method to another. Data traceability is supported by time-stamped raw data, audit trails, user administration and confident certificate of analysis (CoA).

Flexible method parameters for each enzyme type—measuring wavelength, blank measurement, buffer addition, reagent additions, substrate addition, enzyme-specific incubation temperature and time, and data collection duration—allow enzyme assay development and transfer from R&D to quality control laboratories.

And finally, unlike spectrophotometric and microplate methods, a dedicated enzyme analyzer can offer a wide range of incubation temperatures, from 25 oC to 60 oC, expanding the scope of potential applications (3).

For many bio/pharmaceutical manufacturers, there are benefits to be gained from replacing chemicals with enzymes to catalyze reactions. Biocatalysis adheres to green chemistry principles, offering an attractive option for cost-efficient, sustainable manufacturing for processes such as drug synthesis and vaccine development.

An enzyme analyzer based on discrete analyzer technologies offers significant advantages for enzyme research and analysis, including walkaway efficiency combined with faster, easier, and reproducible method development, which leads to better results. These instruments can help fulfill the growing demand for automation and digitization while overcoming the challenges associated with traditional enzyme assays.

The use of accurate, streamlined enzyme activity and kinetics analysis will pave the way for the widespread adoption of biocatalysis in the development of new pharmaceutical drugs, vaccines, and diagnostics kits.

 1 (1–6) 41–55 (2014).
2. S.F. Xu, et al., 

 8 (11) 4208–4211 (2017).
3. M.E. Peterson, et al., 

Hari Narayanan, PhD, is product marketing manager at Thermo Fisher Scientific.

Persistent bottlenecks in biocatalyst development can be alleviated through fully automated enzyme analysis.

Advancing Enzyme Analysis Through Automation

Thermo Fisher Scientific’s new Invitrogen TrueCut Hifi Cas9 Protein is designed for research applications that require highly precise genome editing, such as the engineering of chimeric antigen receptor (CAR)-T cells and the creation of cellular models for disease discovery. Launched in late November 2021, the new protein significantly minimizes off-target events while retaining maximum on-target editing efficiency.

The Invitrogen TrueCut HiFi Cas9 Protein complements Thermo Fisher’s existing portfolio of clustered regularly interspaced short palindromic repeats (CRISPR) genome-editing solutions by minimizing edits in unintended locations, which the company stated in a Nov. 30, 2021 press release are very disruptive in translational research, drug screening, and drug modeling. According to Thermo Fisher, the protein demonstrates near complete elimination of off-target effects compared to wild-type Cas9 in several cell types, including T cells and induced pluripotent stem cells (iPSCs). The company also stated that it has demonstrated improved off-target profiles relative to other products on the market in the release.

"Our pharma and biotech customers are looking for highly precise, accurate solutions to harness the power of CRISPR genome editing," said Brandon Pence, vice-president and general manager of cell biology at Thermo Fisher Scientific, in the press release. "Nothing is more disappointing than to find out months after an edit that the biological response you are measuring was due to an unintended off-target edit. The new Invitrogen TrueCut HiFi Cas9 Protein offers researchers the confidence that their edits took place where they intended, not somewhere else in the genome."

Thermo Fisher Scientific’s Invitrogen TrueCut HiFi Cas9 Protein offers the advantage of significantly minimizing off-target events while aiming to retain maximum on-target editing efficiency.

New High Fidelity CRISPR Cas9 Protein Reduces Off-Target Effects 

OpenEye Scientific and Specifa have jointly developed a software offering known as the OpenEye Orion Antibody Discovery Suite. The first offering from the suite will be AbXtract, an antibody discovery module developed by Specifica.

The Orion Antibody Suite has been described as the only cloud-native. fully integrated software-as-a-service molecular modeling platform, according to a Dec. 2, 2021, press release. The software suite is intended to combine the storage and computational scalability of Amazon Web Services with powerful tools for data sharing, visualization, and analysis in an open development platform.

AbXtract uses proprietary artificial intelligence methods to analyze large amounts of sequence data. The model is designed to help antibody engineers and bioinformaticians uncover diverse antibody sets with ideal properties for downstream therapeutics development.

“What AbXtract does is democratize the use of next-generation sequencing in antibody discovery, letting Orion take care of storage and analysis,” said Andrew Bradbury, co-founder and chief scientific officer of Specifica, in the press release. “Antibody discovery projects that were previously out of reach because of software, hardware, computation, or data sharing limitations have now become possible with AbXtract on Orion.”

New Technology

The partnership between OpenEye Scientific and Specifica has generated AbXtract, an antibody discovery model that is included in the Orion Antibody Suite.

OpenEye Scientific and Specifica Partner to Launch Orion Antibody Discovery Suite

Syngene International, a research, development, and manufacturing services company, announced on Dec. 16, 2021 that the company and Amgen have extended their long-standing multi-discipline research collaboration to the end of 2026.

The scope of the collaboration includes integrated drug discovery and development solutions in discovery chemistry and biology, peptide chemistry, antibody and protein reagents, pharmacokinetics and drug metabolism, and pharmaceutical development. Under the extended contract, Syngene will build and operate a dedicated laboratory, which will enable the acceleration of R&D projects. Syngene currently operates the existing Syngene Amgen R&D Center (SARC) under the companies’ original collaboration deal.

SARC was established in 2016 and was expanded in 2017. The center currently consists of 60,000 ft2 of floor space. A dedicated team of multi-disciplinary Syngene scientists are working closely with Amgen researchers on the discovery and development of innovative medicines.

"While SARC was established in 2016, our collaboration with Amgen dates back to 2012. During this time, we have partnered on research and development to address some of the most serious diseases in the world. We have progressively expanded our areas of collaboration, co-authored research papers on novel science, and supported the advancement of multiple development projects, while adhering to global standards of quality and safety,” said Jonathan Hunt, CEO and managing director, Syngene International, in a company press release.

“Amgen is pleased with the success of the long-standing collaboration with Syngene that has contributed to the advancement of a number of molecules into clinical trials,” said Margaret Chu-Moyer, vice-president of Research and head of Chemistry, Characterization & Technology at Amgen, in the press release. “We look forward to continuing to work together with the shared goal of addressing serious disease for patients in need.”

News

Amgen and Syngene International have extended their research collaboration to 2026.

Amgen Extends Collaboration with Syngene International

Genezen, a cell and gene therapy contract development and manufacturing organization, has officially opened its new process development and analytical lab for viral vector production, the company announced in a Dec. 3, 2021 press release.

The lab includes a full suite of process development capabilities to support current good manufacturing practice (CGMP) and commercial readiness, upstream and downstream process improvements, research-grade and preclinical vector production, and analytical assay development and validation.

This lab is the first phase of a multiphase master plan to build an over 75,000-ft2 CGMP-compliant lentiviral and retroviral vector production facility. The next phase includes CGMP production suites, construction of which is currently underway and due to be complete in early 2022.

When finished, the new facility will conduct aseptic manufacturing of lentiviral and retroviral vectors from multiple CGMP production suites and will offer capabilities for host cell expansion, host cell banking, and viral vector production using transient transfection and producer cell lines. The site will also include analytical testing services, such as recombinant competent lentivirus testing, vector stability testing, and safety and sterility testing. The facility’s end-to-end capabilities will enable Genezen to deliver optimized closed and semi-automated processes for viral vector production.

Genezen will meanwhile continue providing cell manufacturing and patient sample testing through its existing academic partnerships.

“The opening of our dedicated viral vector process and analytical development lab is a key milestone for us in launching our additional early stage capabilities needed to drive the cell and gene therapy industry forward,” said Ray Kaczmarek, CEO of Genezen, in the press release. “Our deep expertise in development and manufacture of viral vectors coupled with this new facility and equipment will accelerate our partners’ abilities to advance life-saving therapies into the clinic.”

Genezen has opened its new process development and analytical lab for viral vector production.

Genezen Starts Up Operations at Lenti- and Retroviral Vector Process Development Lab

Bionique Testing Laboratories, a provider of mycoplasma testing services, has been 100% acquired by a subsidiary of the Japan-based healthcare company, Asahi Kasei Medical. With the acquisition, Bionique will be joined with ViruSure, an Austria-based viral and prion safety testing services company, which is a part of the global biosafety testing services unit of Asahi Kasei Medical’s Bioprocess Division. Both Bionique and ViruSure will continue operations under their existing brands, which Bionique announced in a Dec. 15, 2021 press release.

The acquisition combines Bionique’s mycoplasma and viral safety capabilities with ViruSure’s expertise, expanding Asahi Kasei Medical’s biosafety testing services. Asahi Kasei Medical is also actively investing in facility and employment expansion at various operating sites of both Bionique and ViruSure, according to the press release.

“Joining the Asahi Kasei group will enable Bionique to consolidate its leadership position in the global mycoplasma testing arena and afford our clients access to ViruSure’s service portfolio, satisfying their end-to-end biosafety testing needs from discovery through commercialization,” said Gladis Zamparo, CEO of Bionique, in the press release.

“We are excited to have a strategic partner like Bionique as part of the Asahi Kasei group, and

the expertise in mycoplasma testing that they bring will further strengthen our goal to better

service our customers in all areas of biosafety testing,” said Andy Bailey, CEO of ViruSure, in the press release.

Bionique Testing Laboratories has been acquired by Asahi Kasei Medical and will join the company’s biosafety testing services unit.

Asahi Kasei Medical Acquires Bionique

GlaxoSmithKline (GSK) and the University of Oxford have entered into a major five-year collaboration to establish the Oxford-GSK Institute of Molecular and Computational Medicine, which will be based at the University of Oxford. The institute aims to improve the success and speed of research and development of new medicines by building on insights into human genetics and by using advanced technologies, such as functional genomics and machine learning, GSK announced in a Dec. 2, 2021, press release.

Under the collaboration, GSK will fund Oxford-GSK with £30 million (US$40 million). Scientists from both GSK and Oxford will help prioritize early R&D programs that are most likely to succeed and match them to patients most likely to respond. The Institute will recruit several new research groups. In addition, five GSK/Oxford fellowships will be provided for early to mid-career researchers to establish themselves as principal investigators researching areas aligned with the Institute’s aims and objectives.

The institute will be based in the Nuffield Department of Medicine at Oxford and will be closely associated with colleagues from across departments, including the University’s Welcome Centre for Human Genetics and Big Data Institute. GSK and Oxford are in active discussions on the first projects, which they anticipate starting in the second half of 2022.

The institute will aim to build on the progress made in using genetic evidence to increase the success rates of new treatments in clinical trials and the recent digitization of human biology, a technological advancement that has the potential to improve drug discovery by more closely linking genes to patients. The institute also intends to improve on the understanding of diseases by drawing on recent advances in pathology, which includes methods for measuring changes on a cellular, protein, or tissue level.

As part of its task, Oxford-GSK will evaluate and integrate new approaches in genetics, proteomics, and digital pathology to understand detailed patterns of disease, which vary from individual to individual. The institute will initially focus on neurological diseases, such as Alzheimer’s and Parkinson’s Disease. It will also aim to provide new measurements to establish proof-of-concept for potential medicines earlier in the R&D process by better identifying the most appropriate patients to enroll in clinical trials.

“We are delighted to be joining with the University of Oxford in this new collaboration. By combining the strengths of our two scientific organizations and harnessing advanced technologies, the Oxford-GSK Institute exemplifies the [United Kingdom]’s track-record and continued ambition in life sciences. Together, our aim is to improve drug discovery and development to help bring new and better medicines for patients,” said Emma Walmsley, CEO, GSK, in the press release.

GlaxoSmithKline, together with the University of Oxford, has launched the Oxford-GSK Institute under a new collaboration that aims to study complex diseases to accelerate the success of drug discovery and drug development efforts.

GSK and University of Oxford Partner on Disease Research to Advance Drug Discovery and Development

On Dec. 1, 2021, Roche announced the completion of its share purchase agreement to acquire 100% of the outstanding shares of TIB Molbiol Group in a company press release. The companies will focus on the rapid development of polymerase chain reaction (PCR) assays and diagnostic tests for emerging pathogens and potential health threats, such as infectious diseases. TIB Molbiol will operate as a subsidiary within the Diagnostics division.

For more than 20 years, the companies have collaborated to address biological threats, including:

“I am very pleased that together with TIB Molbiol, we can significantly improve access for patients to a broader spectrum of diagnostic tests—from high volume routine tests to the diagnosis of rare diseases. TIB Molbiol will continue to be an innovation engine and frontrunner in our common fight against infectious diseases, such as SARS-CoV-2,” said Thomas Schinecker, CEO, Roche Diagnostics, in a company press release. “TIB Molbiol has helped to shape the future of PCR infectious disease testing and will continue to drive cutting-edge research so we can bring better healthcare outcomes to patients faster.”

The companies will focus on the rapid development of polymerase chain reaction assays for emerging pathogens and potential health threats, including biological threats.

Pharmaceutical Technology's In the Lab eNewsletter, January 2022