astreabioseparations.com
Table of Contents
Introduction
Introduction. . . . . . . . . . . . . . . . . . . . . . . . 2
Terry Pizzie
Terry Pizzie
Purity By Design. . . . . . . . . . . . . . . . . . . . 3 Marc Hummersone
Downstream Purification Solutions: Addressing Challenges to Cell and Gene Therapy Manufacturing. . . . . . 4 Daniella Steel
FPLC Column Selection Considerations. . . . . . . . . . . . . . . . . . . . . . 6 with Dan Yukon
Alternatives for Affinity Adsorbent Development in Downstream Bioprocessing . . . . . . . . . . . . . . . . . . . . . . 8 Bruce Dawson
Bolstering Manufacturing Capacity and Achieving Supply-Chain Resilience. . . . . . . . . . . . . . . . . . . . . . . . . 11 with Ari Ojinaka
T
hank you for spending some time with Astrea Bioseparations today. I joined the team in the middle of 2021 after an eventful year. In 2020, the company took the name Astrea Bioseparations and began brand elevation work to reflect its new direction. Then, as we all know, the COVID-19 pandemic took over daily life. Through it all, I am proud to have seen how the Astrea Bioseparations team has reacted. We have remained focused on serving customers, supporting industry advancement, and doing the work needed to secure our position as a leading bioseparations solutions provider. We have made key investments throughout this year, and such efforts will continue in 2022. Most notably, we expanded research and development capabilities in Cambridge, UK, and registered a new branch office in Singapore to support customers in the Asia–Pacific region. We have also expanded our world-class talent pool in addition to aligning resources for multiple innovation-centered collaborations in the biopharmaceutical industry. As the team pulled this report together and reflected upon 2021, it was difficult not to reflect on what an honor it is to work in our industry. Walking into 2022, we wish all our colleagues a safe, happy year, and we appreciate your joining us as the journey continues. c Terry Pizzie is chief executive officer of Astrea Bioseparations, Ltd., The Freeport, Ballasalla, Isle of Man, IM9 2AP, United Kingdom; https://www. astreabioseparations. com.
2
BioProcess International
20(1–2)si
January–February 2022
Sponsored
Purity By Design Marc Hummersone
A
strea Bioseparations has a well-established modular program to support customer work from small to large scales with ligands, adsorbents, and chromatography columns that design purity into each process. Demand for increased productivity in biopharmaceutical manufacturing has placed new pressure on downstream purification operations. For recombinant proteins and monoclonal antibodies (MAbs), such pressure stems from significant gains in upstream productivity, particularly from high titers produced using increasingly efficient cell-culture systems. However, for viral vectors used in gene and genemodified cell therapies and viral-vector vaccines, pressure arises from growing demand for complex products that currently are produced at low titers. Harvested feedstocks are consequently complex and typically have high levels of process- and productrelated contaminants, many of which are structurally similar to target molecules. Thus, many steps are required to achieve desired purity levels for target capsids. But during each such step, some target viral vectors are removed along with the impurities. There is great need for solutions that increase yields of full viral-vector particles to reduce the need for extensive purification. In the meantime, methods that improve purification of low-titer process fluids will be essential to reducing the footprint, cost, and timelines involved in manufacturing therapies and vaccines based on viral vectors. Astrea Bioseparations addresses downstream inefficiencies with products and services that can shorten development timelines and increase the efficiency of subsequent chromatography and other purification operations. We provide single-use and refreshable chromatography solutions to mitigate the need for cleaning and validation processes when setting up new purification runs. Designed for campaign use, our prepacked, disposable columns are easy to install, helping users to reduce turnaround times between product runs and improve process economics generally. Columns can be packed with any resin on the market, which reduces needs for on-site packing skills and column storage. In addition to offering a broad range of off-theshelf catalog ligands for affinity chromatography, Astrea Bioseparations provides solutions for customers Sponsored
working with novel biomolecules such as viral vectors for cell and gene therapies. We develop our own solutions and help customers to optimize their own processes by building “purity by design” into the process. Ligands can be tailored to meet specific separation needs and developed with efficient adsorbents through to commercial scale. It is important to ensure that selected ligands address an application’s requirements. Lead candidates can be optimized with adsorbents and columns that enable effective processing. Automated chromatography workstations and prepacked columns can accelerate an optimization process, which includes initial investigation of nonbound, wash, elution, and clean-in-place steps. Adsorbent development involves evaluation of base matrices, ligand synthesis methods, and ligand immobilization approaches, including attachment chemistries and ligand densities. Chromatography development work includes identification of process conditions that optimize adsorbent use and maximize product purity and yield. Typically, these two development steps are performed in parallel. Once testing has identified an ideal ligand and adsorbent, the next step is delivery. This includes technology transfer and commercial, large-scale manufacture of a custom adsorbent at our cleanroom manufacturing facilities based on the Isle of Man, UK. Different batch sizes of affinity adsorbents can be accommodated, including single batches <1,000 L. Good manufacturing practice (GMP) standards have been adopted during absorbent production to support our customers with a full regulatory package. If desired, we can provide GMP-ready prepacked columns containing custom adsorbent. c
Achieving Purity By Design Astrea Bioseparations is honored to support the biopharmaceutical industry with products and services that help bring life-saving and -improving medicines to market. Regardless of process scale, we are ready to build purity by design into each process to ensure quality and speed to market. Marc Hummersone is senior director of research and development at Astrea Bioseparations, Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, United Kingdom.
January–February 2022
20(1–2)si
BioProcess International
3
Downstream Purification Solutions Addressing Challenges to Cell and Gene Therapy Manufacturing Daniella Steel
I
nterest in cell and gene therapies (CGTs) remains high, with significant and ongoing venture capital investment driving continued growth in the advanced therapies sector. Numerous novel treatments are entering preclinical development and progressing through clinical trials, increasing demands on biomanufacturing capabilities. Many of those candidates target larger populations and require larger doses than have previous generations of CGTs, placing further pressure on biomanufacturers. Simultaneously, the biosimilars market continues to grow as more biologic drugs lose patent protection. Biosimilars could be beneficial because they are more cost-effective than their originator products, they can be produced in about half the time, and their market acceptance and uptake have already been established. Uptake is expected to be particularly strong in Brazil, India, and China. Biopharmaceutical manufacturers are responding to such trends by expanding their capacity, often by incorporating single-use bioreactors with volumes >2,000 L. Contract manufacturing organizations (CMOs) also are increasing the flexibility of their facilities and diversifying their capabilities to support multiple modalities, such as monoclonal antibodies (MAbs) and therapies that leverage adenoassociated viral and lentiviral vectors. Ongoing technological advances — e.g., improved design by directed evolution of viral vector capsids for gene therapy — are enhancing biopharmaceuticals steadily. Implementation of such solutions enables development of next-generation vectors with improved abilities to deliver genetic cargo to target organs and tissues. However, rapid technological advances can create new problems for process development. Supply-chain issues surrounding consumables such as chromatography media and single-use systems are creating additional problems for drug manufacturers. Shortages stemming from rapid growth in the CGT sector and complications from the COVID-19 pandemic have highlighted the risk of 4
BioProcess International
20(1–2)si
disruption that comes with reliance on a single supplier and thus have encouraged drug makers to refocus on ensuring supply-chain integrity and redundancy. Despite the difficulties of securing multiple sources, biomanufacturers have been faced with the need to qualify secondary suppliers and run comparison studies on substitute consumables for contingency plans.
Downstream Bottlenecks
Improvements to biomanufacturing efficiency and productivity have significantly increased upstream production volumes and titers for traditional engineered proteins and antibodies. Downstream throughput and capacity must increase accordingly, a need that has driven interest in downstream process intensification to increase productivity within small footprints. Meanwhile, growing diversity among biological treatment modalities is complicating the development of a downstream purification platform approach that would improve process economics considerably, such as what was established by introducing protein A purification for MAbs. In fact, many other downstream processes leverage legacy technologies that were designed for MAbs. Such solutions do not accommodate the unique requirements of novel modalities such as viral vectors and exosomes. CGT purification is more complex still, requiring additional steps and workflows. And despite high expression titers upstream, feedstream concentrations of many therapeutic drug substances remain low, with high contaminant-to-target ratios. Furthermore, genomic DNA and chromatin contaminants can foul chromatography media easily, and large viral vectors often cannot access the inner surfaces of traditional chromatography resin beads. In the latter case, slow and suboptimal capture steps result when most of a resin’s binding surface area goes unused. Current downstream processes can create unfavorable conditions — e.g., extreme pH and shear
January–February 2022
Sponsored
forces — that deteriorate the quality of a biological drug substance, diminishing yields and generating less-than-desirable product quality. The ultimate result is increased costs for patients.
Fit-for-Purpose Tools
Validating multiple suppliers for key raw materials and other consumables has become essential to ensuring their robust supply and enabling continuous production of advanced therapies. Regulatory agencies have not provided explicit instructions regarding the demonstration of comparability for products made using materials from different suppliers, however. As well as introducing uncertainty, this new validation process adds time and cost to an overall development process. Chromatography steps within downstream purification show some potential for increased productivity when using a fit-for-purpose approach. For instance, a key consideration during viral vector purification is removal of capsids that are empty or packed with the incorrect generic material. Such impurities can have a direct impact on final product quality. Consequently, chromatography solutions that separate full capsids, or at least enrich them, are actively being developed. Use of affinity chromatography during a capture step can reduce the overall number of unit operations that are required in a downstream process, thereby increasing efficiency and reducing cost. Multicolumn continuous chromatography, on the other hand, can improve use of resin capacity and increase process productivity in a reduced footprint and with less waste. The Astrea Bioseparations portfolio includes offthe-shelf and custom synthetic ligands and chromatography resins for purification of specific biomolecules. A license to the Avacta Life Sciences Affimer (stefin A) platform expands upon proven Mimetic Ligand chemical ligand libraries to expand the range of ligand discovery and development capabilities. Affimer proteins can be designed for exquisite specificity and are critical to powerful platform ligand discovery.
Expanding Access Through Innovation
Astrea Bioseparations is developing nanofiber technology as an innovative chromatography matrix. Composite electrospun cellulose nanofibers of uniform composition are functionalized physically or chemically for different separation modalities, including ion-exchange, hydrophobic-interaction, affinity, and steric-exclusion chromatography Sponsored
mechanisms. Rapid binding kinetics and residence times shorter than one second allow high flow rates even at low pressures. High-capacity separations are made possible in a small footprint with reduced processing times and costs with applications from laboratory to clinical scales.
A Reliable Platform
The Astrea Bioseparations portfolio also features an internally designed and manufactured range of PuraBead near-monodisperse agarose chromatography beads. They are produced within a narrow size distribution for optimal flow properties, and they are available for different separation modalities, including high-performance anionexchange and hydrophobic-interaction chromatographies for bioprocessing contaminant removal or target protein capture. Our EtoxiClear product provides cost-effective and efficient endotoxin removal using a highperformance synthetic-ligand affinitychromatography adsorbent. With a high dynamic binding capacity for endotoxin and low protein binding, the product can achieve superior removal of endotoxins generated during bioprocessing with product recoveries >90%. As well as internal and customer projects, the team at Astrea Bioseparations is collaborating with companies across the biopharmaceutical industry to drive innovation and bolster interest and education in this evolving industry. For instance, we are enhancing our nanofiber chromatography solutions in conjunction with Nanopareil, which developed the technology initially, and VectorBuilder, which is running proof-of-concept tests for purification of viral vectors.
Expanding Capabilities and Reach
The Astrea Bioseparations team is committed to supporting developers of cell and gene therapies, conventional biologics, and biosimilars with improved downstream processes that maximize yields. Whether through off-the-shelf purification technologies or tailored chromatography solutions (including prepacked columns), the team is focused on helping customers and partners accelerate development timelines with a “right the first time,” fit-for-purpose approach that optimizes an end user’s process. c Daniella Steel is senior product manager for cell and gene therapy applications at Astrea Bioseparations Ltd., Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, United Kingdom.
January–February 2022
20(1–2)si
BioProcess International
5
Fast-Protein Liquid Chromatography Column Selection Considerations BPI Staff with Dan Yukon
O
n 10 November 2020, BPI presented an “Ask the Expert” webinar with Dan Yukon (head of North American and global SNAP product sales at Astrea Bioseparations) on considerations for selecting analytical fast-protein liquid chromatography (FPLC) columns. With many options on the market, deciding which type and brand to use can be difficult. To help take out the guesswork, Yukon addressed pressure and volume considerations; column configuration; materials of construction; frit type, design porosity, and mounting; connection types; adjustability; construction accuracy; packing tube design; and column storage.
Yukon’s Presentation
Speaking from a mechanical engineering background, Yukon explored considerations for column selection, assuming low-pressure applications, use of glass or plastic columns, and manual packing by users. Volume — of both resin and sample — is the core starting point, Yukon stated. Different resins capture different sample volumes. Once you understand what you’re trying to collect and how much, then you know which resin family to begin with. Suppliers can offer guidance from that point. Aspect ratio is the ratio of a column’s diameter to its length. For a given volume, you can use a tall, slim column or a short, fat one, the latter allowing for higher flow rates and shorter residence times. Which aspect ratio you need is easy to identify. In size-exclusion applications, for example, samples must have enough time to interact with the chromatography resin. Resin suppliers can suggest the best configuration of their columns for specific applications. Flow distribution also is important. Tall, thin columns generate more wall effects; wide columns with relatively short bed heights need good flow distribution from an inlet connection, typically in a top-center location. You don’t want flow moving only through the center of the bed. Pressure Considerations: Users should consider carefully the expected backpressure generated by a packed resin bed. FPLC resin beads normally come in sizes of 30–90 µm, making them compatible with 6
BioProcess International
20(1–2)si
most column brands. High-performance resin beads are even smaller (e.g., 20 µm), which can cause significant backpressure in use, creating challenges for glass and plastic columns. Column ruptures from excessive pressure can be dangerous and waste both resin and sample. The larger the resin particle size, the less that backpressure is a concern. Materials of Construction: Glass is more fragile and brittle than plastic and tends to fail dynamically; plastic typically will crack to relieve pressure. Column materials should be reviewed for compatibility with all slurry, packing, testing, storage, and cleaning buffers and reagents for all wetted components, including seals, frits, flow adapters, and connectors. Yukon also recommended consideration of temperature during such evaluations. Connections and Frits: Most manufacturers of FPLC systems have standardized their connections to system manifolds to 1/16 inch for small columns or 1/8 inch for large ones. Tubing to and from a column to a detector should be minimized to prevent sample diffusion. Available in a range of materials, porosities, and pore structures, frits often are overlooked and misunderstood. Their function is to facilitate fluid pass-through while holding back chromatography media. Frit porosity should never exceed half the diameter of the resin particle (e.g., ≤20-µm frit pores for a 40-µm resin). But you don’t want to restrict flow through the column. Users should consider the average particle size of their resin as well as the fines content to achieve the best column performance.
January–February 2022
Sponsored
Frits can be a source of flow hang-up, restriction, and dead volume. Flow rates and backpressure figure into choices made here. Polyethylene is a widely accepted material, but more aggressive solvents can require stainless-steel, sintered-glass, or even titanium frits. Consider frits and O-rings to be consumable/disposable and replaceable components. They should be changed out rather than cleaned every time that a column is repacked. Testing and Qualification: Successful chromatography operations require a fully tested and qualified column. Asymmetry values and theoretical plates are the standard measurements — often tested with a concentrated injection of NaCl or acetone solution. Yukon said that users must understand the interrelationships among injection volume, overall column/bed volume, and flow rate. The most popular detection method for these evaluations is conductivity. If a column does not meet your qualification standard, then chromatography results will be compromised or inaccurate. Your resin’s slurry mix needs to be correct, as do the flow rate, compression factor, and evaluation criteria. Price and Support: Price always is a consideration in selecting FPLC columns. They come in a broad range of configurations, construction materials, pressure ratings, and technical ability — with corresponding price differentials. For academic training and cases for which performance is not an issue, simple gravity-fed columns suffice. They are relatively safe to use and cannot develop dangerous backpressures if misused by inexperienced users. For most bench scientists, however, column cost is relatively insignificant compared with the cost of sample materials. Reliability and reproducible results are important, so hardware should be selected with performance in mind. A column’s performance is only as good as its packing. Users should consult with resin manufacturers about the best way to pack a given resin. Most media need to be slurry packed, for which proper technique and equipment are required. Users also should consult with column manufacturers. Scale-Up: During process development, it is incumbent on scientists doing small-scale purification to respect and understand the challenges facing the group that will take the process to the next level. Yukon advised consideration of the “big picture” in anticipation of purification at larger scales. Developing familiarity with a type of column helps. Doing that enables understanding of challenges involved in packing and configuring those columns. Sponsored
Just selecting a column with a higher initial pressure tolerance than you think you need indicates that the column is built robustly and will be durable in the long run while offering flexibility in the laboratory. Columns with high pressure tolerance generally can be used at lower pressures. Those with interchangeable components provide further flexibility for future projects. Yukon recommended finding out what maximum size is offered in a given product family. Yukon added that users must evaluate column suppliers carefully, with a clear understanding of their stock, lead times, configurations, and accessory availability. Columns with good, repeatable performance and broad availability should be specified in method development to maintain continuity with specifications. When evaluating columns for a particular application, consider current and future needs. Interchangeability is important.
Questions and Answers What are the most common “rookie mistakes” made when selecting or using chromatography columns?
Particularly in academia and small laboratories, a scientist is tasked with performing a purification, and that person grabs the first column that looks close to what is needed. The scientist tries to pack it but doesn’t truly understand its parameters and specifications, maybe assuming a column’s diameter and diametrical tolerance. That person might not even change the frits or understand that those left over from a previous experiment are inappropriate for a new application. Novices may also spend a lot of time preparing the resin and packing the column, then get a poor result. That’s when we get a call. The best thing to do is to understand your hardware and application requirements. Then proceed methodically.
What are good resources to consider when selecting columns? Suppliers are helpful resources. If you have a
resin in mind, then contact the manufacturer about hardware recommendations. Especially if you are buying bulk resin and packing your own columns, you should know whether a supplier can provide application data before you get started. Also develop a relationship with your column supplier. It helps to have a three-way dialogue: scientist, column manufacturer, and resin supplier. Astrea Bioseparations often participates in such conversations. Our team can make recommendations and head off problems before the work begins.
Watch the presentation at https://bioprocessintl.com/sponsoredcontent/fplc-column-selection-considerations. c
January–February 2022
20(1–2)si
BioProcess International
7
Alternatives for Affinity Adsorbent Development in Downstream Bioprocessing Bruce Dawson
A
successful affinity adsorbent features exquisite selectivity, reversible binding, and high binding capacity — in addition to being robust, resistant to fouling, affordable, and scalable. However, such qualities differ based on protein of interest, feedstock, and upstream process conditions. Because the probability of a high-quality hit during ligand screening depends on the number of candidates that are screened, it is important to have access to a large library of potential ligands with different properties. Astrea Bioseparations leverages two ligand discovery platforms. Our CCL synthetic chemical
combinatorial library is diverse, comprising 100,000+ triazine-based ligands. They are highly stable, nontoxic, and nonmutagenic. The associated adsorbents are inexpensive, scale up easily, and have strong safety profiles, as demonstrated by an excellent track record in regulated downstream processes. Alternatively, Affimer ligands (developed by Avacta and licensed by Astrea Bioseparations) show strong selectivity, good chemical and thermal stability, broad tolerance to organic solvents, and applicability across a wide range of pH values. This library is also highly diverse, with 1010 ligands.
Figure 1: Phases in development of a good manufacturing practice (GMP)–ready affinity chromatography adsorbent
Phase 1
Phase 2
Ligand Discovery
Adsorbent and Process Development
Technology Transfer and Validation
Regulatory Support
Ligand Design
Adsorbent Development/Optimization • Base matrices • Ligand density • Spacer arms and attachment chemistry • Ligand synthesis method
Three Verification Batches
Extractables and Leachables
One TechnologyTransfer Batch
Stability
Three Validation Batches
Toxicology
Ligand Synthesis Ligand Screening Verification Chromatography
Chromatography Development/Optimization • Performance assessment • Load, elution, and wash conditions • Cleaning-in-place conditions • Initial evaluation of reuse
Figure 2: Different pathways can be taken during discovery of synthetic affinity ligands. (vHTS = virtual high-throughput screening) Target protein structure known and available? Yes No Binding site known? Yes Docking and/or vHTS
8
No
Ligand/inhibitors known? Yes
No
Binding-site Diverse Similarity or determination library pharmacophore or blind design searches docking and HTS
BioProcess International
20(1–2)si
January–February 2022
Phase 3
Phase 4
Because the probability of a highquality hit during ligand screening depends on the number of candidates that are screened, it is important to have access to a LARGE LIBRARY of potential ligands with different properties.
Sponsored
Development of a good manufacturing practice (GMP)–ready affinity chromatography adsorbent comprises four modular phases: ligand discovery, adsorbent and process development, technology transfer and validation, and regulatory support (Figure 1). Here, I describe the first two phases using our CCL and Affimer ligand platforms.
From Ligand Discovery to Manufacturing Using the CCL Synthetic Ligand Library
Several paths can be followed during discovery of synthetic affinity ligands (Figure 2). Which route to take depends on what information is known — and available to analysts — about a target protein’s structure, binding sites, ligands, and inhibitors. Computational techniques can be applied at several stages of a ligand design process. Such methods also are used to enhance process engineers’ understanding of a target protein and its adsorbent interactions. Initial Evaluation of Ligand Performance: In a case representative of the ligand-screening process, Figure 3: Purification performance of a lead ligand candidate for the second customer project
Absorbance (AU)
Target protein
Absorbance (AU)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0
Target protein
2
4
6
Table 2: Purification performance of a customer’s lead ligand candidate at pilot scale after adsorbent and chromatography development (BC = binding capacity, HMW = species of high molecular weight)
Load Total protein titer: 1.8 mg/mL Target protein titer: 0.7 mg/mL Purity: 39%
Lead adsorbent Binding Capacity: 17 mg/mL Recovery: 96% Purity: 90%
8 10 12 14 16 18 20 22 24 26 28
Time (minutes)
Table 1: Results from secondary screening of four candidate ligands for the second customer project; candidate 2 underwent further study and optimization during adsorbent and chromatography development. (BC = binding capacity, HMW = species of high molecular weight, expressed as a percentage of the purified drug substance) Ligand
BC
Yield
Purity
HMW
Dimers
1
2.4 g/L
56%
81%
0.5%
15.2%
2
3.1 g/L
68%
82%
0%
10.0%
3
2.3 g/L
73%
75%
3.0%
19.9%
4
2.1 g/L
55%
89%
0%
11.8%
Sponsored
Astrea Bioseparations helped a customer to select a synthetic affinity ligand for purification of a biomolecule. After primary screening of 768 potential ligands and secondary testing of 29 candidates, three ligands advanced to additional study of purification performance in pilot-scale conditions. At the end of this process, a lead candidate was selected based on its potential for significant gains in target-protein purity and recovery. Subsequent Process Development: We worked with another customer to identify suitable ligands for purification of a different protein. During secondary screening of four candidates, ligand 2 showed the most promising profile (Table 1). For that candidate, we performed adsorbent and chromatography process development to examine the influence of factors such as ligand densities, attachment chemistries (e.g., spacer arms), and process conditions (Figure 3). Such studies enabled optimization of the candidate ligand and adsorbent to maximize product purity and yield. Table 2 lists results from this development work.
BC (g/L)
Yield
Purity
HMW
Dimers
Lead Candidate
16
>95%
92%
0%
<5%
Rate of Improvement
>5-fold increase
+25%
+10%
—
50% decrease
Table 3: Performance of an Affimer ligand candidate immobilized onto a PuraBead P6HF adsorbent for purification of factor IX; results were obtained by analyzing fractions from triplicate chromatography runs. Specific Activity (IU/mg total protein)
Purity (fold increase)
Load
Elution
—
Average
2.71
156.23
57.55
Standard Dilution
0.08
9.83
2.00
Factor IX Activity (IU/mg protein)
Active Factor IX Recovery (%)
Load
Elution
—
Average
134.29
120.69
89.88
Standard Dilution
2.08
5.99
4.26
January–February 2022
20(1–2)si
BioProcess International
9
Figure 4: Development of an Affimer ligand for human factor IX, a treatment used in hemophilia; factor IX was the target for phage display selection. Factors II and VII served as deselection targets for phage display. Target: Factor IX
Target protein quality control
Primary screening
All clones were assayed in the iQue primary screen.
Deselection targets: Factors II and VII; 167 clones were selected.
Phage display
Sequencing
42 positive clones were sequenced.
Subcloning and colony picking
Subcloning and expression
5 Affimer ligands were subcloned.
Small-scale expression
Pull-down and characterization
Lead Affimer ligand was selected.
Figure 5: SDS-PAGE results from triplicate chromatography studies of an optimized Affimer ligand; runs listed in black, red, and green represent load, nonbound proteins, and proteins from elution fractions, respectively. The target protein (human factor IX) is marked with purple arrows.
Figure 6: Breakthrough capacity of an optimized Affimer ligand immobilized to a PuraBead P6HF adsorbent for purification of human factor IX 0.35 0.30 0.25
C/C0
0.20 0.15 0.10
0.05 0.00 0
Affimer Ligand Discovery and Adsorbent Development
BioProcess International
20(1–2)si
20
30
40
50
Cumulative Volume (mL)
60
70
(BCA) total-protein assays. Results demonstrated 90% recovery of purified factor IX with a 58-fold increase in specific activity. Minimal optimization was required to achieve these results. Breakthrough Capacity: Binding capacity at 10% was 2.65 milligrams per milliliter of adsorbent. The sharp curve was typical for an affinity adsorbent.
The Astrea Bioseparations team worked with a customer to identify and develop an Affimer affinity ligand for purification of human factor IX, a serine protease that can be produced and administered as a treatment for hemophilia B. Factor IX served as the target for phage display selection; the deselection targets for phage display were factors II and VII. Purification Performance: A lead Affimer candidate was immobilized onto PuraBead P6HF hydrophobic-interaction chromatography (HIC) adsorbent. Triplicate chromatography runs were performed using a factor-IX–containing feedstock. Fractions were analyzed using coagulometric, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), homogeneous timeresolved fluorescence (HTRF), and bicinchoninic acid 10
10
Meeting Diverse Ligand Criteria
Using multiple ligand discovery platforms facilitates development of affinity adsorbents for many different kinds of target molecules from complex feedstocks. The CCL synthetic-ligand and Affimer library technologies provide different advantages that can be exploited to meet diverse ligand criteria. c Bruce Dawson is a customer services field expert with Astrea Bioseparations Ltd., Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, United Kingdom. Affimer is a registered trademark of Avacta Life Sciences Ltd. CCL and PuraBead are registered trademarks of Astrea Bioseparations Ltd.
January–February 2022
Sponsored
Bolstering Manufacturing Capacity and Achieving Supply-Chain Resilience BPI Staff with Ari Ojinaka
T
he COVID-19 pandemic has exposed vulnerabilities in biopharmaceutical-industry approaches to supply-chain management. Drug manufacturers have tended to presume that their suppliers have access to raw materials for bioprocess components. But global crises can disrupt reliable access. Ari Ojinaka (production manager at Astrea Bioseparations) joined BPI in October 2021 to explore strategies for navigating supply-chain uncertainty. He described how his company optimizes capacity, enhances communication with its suppliers and customers, and supports a quality-driven culture.
Ojinaka’s Presentation
Traditional supply-chain strategies remain relevant, Ojinaka explained. But focusing on supply-chain reliability cannot mitigate significant disruptions posed by events such as Brexit and the COVID-19 pandemic. Striving for supply-chain resilience helps to manage changing economic conditions. Resilience depends on visibility, which describes a company’s ability to anticipate disruptions and respond in ways that minimize impacts to product delivery. Improving supply-chain visibility includes enhancing communication with customers and rawmaterials suppliers to hone supply-and-demand planning. Robust data analytics can help companies to understand economic trends and plan accordingly. Resilience also requires agility. Building capacity redundancy enables quick reactions to short-term changes, yet operations must be efficient. Suppliers must balance cost of materials against availability of resources that could accommodate demand shifts and minimize impacts from delayed shipments. Ojinaka described his company’s response to a COVID-related supply-chain disruption. Early in 2021, a supplier contacted the Astrea Bioseparations facility on the Isle of Man, UK, about lockdown-related rawmaterial shipment delays. A second supplier faced similar obstacles. That material was required to manufacture a product for a customer in 10 weeks. Clear and frequent communication with suppliers enabled the Isle of Man team to address the problem quickly. Management worked closely with customers to notify them about potential delays and supplier Sponsored
changes. Redundant capacity enabled the team to adjust operations to accommodate a new timeline. The site also benefited from flexible workspaces and previous opportunities for cross-training. The site’s quick response stemmed in part from its previous implementation of a three-stage quality risk management (QRM) approach to supplier changes. A quality assurance (QA) team vets available suppliers during sourcing and qualification. Material specifications and criticality assessments help the team to determine how much and what kind of work must occur during qualification. Quality-impact review includes risk assessment, an official change-control request (CCR), and evaluation of trial batches. Implementation entails completion of a CCR, addition of a qualified supplier to the Astrea Bioseparations material and audit system, and updates to documentation. Before the pandemic, the company notified customers about supplier changes during quality impact review. Now, it does so as early as possible to increase transparency and strengthen communication. Astrea Bioseparations plans to bolster its supplychain resilience. It has developed internal capabilities for manufacturing affinity ligands, base matrices, and attachment chemistries; invested in data orchestration systems to preempt supply-chain fluctuations; and worked with customers to establish safety stocks. Agility could be increased by upskilling employees. As evidenced by a new site in Singapore, the company also is expanding its distribution networks. c
Questions and Answers How much capacity does Astrea Bioseparations set aside? The company generally uses 75% of available manufacturing capacity, which provides enough resources to accommodate additional projects.
How long does it take to qualify a new supplier? A week
or two might be enough to evaluate suppliers of noncritical raw materials. Critical materials necessitate extensive testing of trial batches. That process could take 10 weeks.
Watch the presentation at https://bioprocessintl.com/sponsoredcontent/manufacturing-capacity-and-supply-chain-resilience.
January–February 2022
20(1–2)si
BioProcess International
11
Committed to inclusion, innovation, and quality Isle of Man | Cambridge (UK) | Massachusetts (US)
astreabioseparations.com