Figure 1.
This study describes a simple procedure for improving insect cell yields in a benchtop cell culture bioreactor. Here, yields of
Spodoptera frugiperda
(Sf-9) cells were increased by nearly 29% through monitoring dissolved carbon dioxide (dCO
2
) levels in the culture and adding air to the vessel headspace to reduce dCO
2
. The method can also be used to maximize yields in a wide range of mammalian cell types.
Introduction
Producing high yields of protein from insect cells usually requires maintaining high levels of dissolved oxygen (DO) in the culture. However, as cell concentrations grow, they generate an ever-increasing level of dCO
2′
which can inhibit cell growth. We compared growth of Sf-9 insect cell yields in two runs, first without control of dCO
2
levels and then by continuously flowing air into the vessel headspace above the liquid media level to reduce dCO
2
concentration. This insect cell culture protocol has not been fully optimized to obtain the highest yields possible — but it...
+1Photo 1.
Demand for single-use bioprocess systems in the biotech and pharmaceutical industry has increased significantly in recent years. Among other reasons — such as operations, sourcing, and validation — their lower initial investment capital makes disposable systems very attractive for the pharmaceutical industry.
When Wave Biotech introduced the first wave-action reactor/mixer, it was seen by some as the start of single-use technology. Once containers become single-use, all other parts of the manufacturing process ideally have to follow the same principle. These include connectors, tubings, pumps, ultrafiltration/diafiltration (UF/DF) devices, and last but not least: sensors.
Whereas the use of plastic containments is a fairly new development for manufacturing processes, in diagnostics it has been common for decades. The first microplates were made by PMMA in the early 1950s. PreSens offers pH and oxygen sensors for the whole choice of disposable systems ranging from microplates to tubes and bags.
Se...
Figure 1.
CellPrime rTransferrin recombinant human transferrin provides an animal-free alternative to iron salts for industrial cell culture. It is a recombinant analogue of human transferrin expressed in
Saccharomyces cerevisiae
. Supplied as a human holo-transferrin analogue, CellPrime rTransferrin binds specifically to the transferrin receptor, thereby facilitating iron uptake into cells for optimal cell culture performance. Here we describe cell culture data demonstrating that CellPrime rTransferrin shows equivalence to human transferrin (hTf) and superiority to bovine transferrin (bTf) in stimulating cell growth and protein production across a number of industrially relevant cell lines.
Results
Human Recombinant and Native Transferrins Promote Greater Growth and Protein Production than Bovine Transferrin:
To compare the efficacy of CellPrime rTransferrin to commonly used transferrin media supplements, it was compared with serum-derived human transferrin (hTf) and bovine transferrin (bTf) at identic...
+4 Figure 1.
How well does absorbance correlate with cell density and viability measurements in batch and fed-batch cell culture? As expected in science and nature, the answer depends on a set of factors.
Many people working with batch or fed-batch cell culture will reference discrete offline measures of cell activity. Total cell density (TCD), viable cell density (VCD), and percent viability can be evaluated offline using a hemocytometer and accepted trypan blue exclusion method. Cedex® and Vi-CELL™ are two examples of automated, offline cell counters.
With introduction of inline NIR probes used to monitor cell culture, characterization is provided in real time. A common initiative is to correlate inline measurement with offline results to eliminate duplication and improve controlled response time. If results are viewed as a collection over the entire run, data correlations may appear baffling. Relationships over a complete cell culture run need to be segmented depending on certain conditions.
Condition 1: ...
The development of single-use bioreactor technologies is focused on bringing new designs onto the market for effective oxygen transfer and cultivation of not only cell culture processes, but also microbial cultures. The advantages of disposable technologies are: ease of use, prevention of cross-contamination, labor and capital investment savings, reduced qualification and validation, and increased flexibility.
The costs of disposables are lower compared with conventional stainless-steel bioreactors because the capital investment is lower. Therefore, the need is reduced for qualification and validation processes, as well as maintenance. Most single-use bioreactor systems are supplied with reusable sensors that are to be aseptically inserted. This involves risky and time-consuming tasks such as sterilization, calibration, and insertion of the sensors into the bioreactor.
Ideally, single-use bioreactor systems should use disposable sensors. The BIOSTAT® CultiBag RM is a disposable bioreactor that operates by...
Whether applied to business security, product specification, financial exposure or personnel safety, quality is a key element underpinning any biopharmaceutical risk mitigation strategy. A quality system should not be inherently dependent upon personnel, nor should it rely solely on people to ensure the quality of the products manufactured. Irrespective of human resources, the system itself should provide consistency, addressing risk from design inception through to product delivery, ensuring that robust processes are in place that minimize risk to the product and ultimately the end user.
Industry Demands Increased Product Quality
Although not all products supplied to the biopharmaceutical industry are regulated or licensed by a governmental agency, the sector rightly expects an equivalent level of controls, systems and compliance — increasingly to CGMP standards. As a leading global supplier of cell culture media, sera and buffers, SAFC Biosciences understands this requirement, providing specified materi...
Cook Pharmica is a full-service biopharmaceutical contract manufacturer located in Bloomington, IN. Cook Pharmica’s mission is to provide process development, flexible manufacturing, and comprehensive analytical services to the global biotech community; enabling its partners to bring their life-saving discoveries to patients. And with the recent announcement of its expansion into parenteral contract manufacturing, Cook Pharmica will serve the industry as a complete one-source solution.
The current API manufacturing facility was designed to support clinical and small-scale commercial products and features two 2,500-L stainless steel bioreactors. The facility’s design incorporates disposable technologies, segregated production rooms, innovative wall pass-throughs, and unidirectional flow of operators, and equipment and supplies to reduce the risk of contamination. An additional small-scale CGMP API manufacturing train provides up to 250 L capacity in disposable bioreactors.
Cook Pharmica’s development group...
Figure 1.
Antibody-based drugs represent a growing segment of the pharmaceutical industry and one of the most promising classes of therapeutic drugs. The demand for protein-based drugs is expected to grow steadily for the foreseeable future. However, production of monoclonal antibody (MAb)–based drugs remains very costly, and solutions to lower production costs are needed. Downstream purification steps are becoming a target for cost reduction. The use of ion-exchange chromatography to directly capture MAbs from cell culture streams or as a polishing step after an affinity separation may be a way to reduce costs.
Ion-exchange chromatography is an established step in the purification of MAbs following protein A capture. In this application note, we demonstrate the utility of the combination of a cation exchange step and an anion-exchange step using UNOsphere Rapid S media and UNOsphere Q media (Bio-Rad Laboratories, Inc.), respectively. The mirrored binding characteristics of UNOsphere Rapid S and UNOsphere...
Figure 1.
Monoclonal antibodies (MAbs) are expected to dominate the future biopharmaceutical landscape (
1
). Although chromatography is the mainstay of MAb downstream purification processes, more efficient and cost-effective chromatography technologies are needed to address increasing MAb titers in mammalian cell culture supernatants.
Ion-exchange membrane chromatography has been demonstrated to capture large biomolecules such as viruses and plasmid DNA with higher dynamic binding capacity than ion-exchange beaded column chromatography (
2
,
3
). Recently, anion-exchange membrane chromatography has been viewed as a viable alternative to column chromatography for trace contaminant removal from a protein A-purified MAb (
4
,
5
).
Here, a 0.35-mL membrane volume (MV) Mustang® Q coin was evaluated for Chinese hamster ovary (CHO) host-cell protein (HCP) and host-cell DNA removal from a protein A purified MAb. Spiking a protein A-purified MAb with CHO DNA showed more than 4.9-log removal at pH 8.0 and 4 mS/c...
Figure 1.
Tangential-flow filtration (TFF) cassette operations are known for reusable cassettes; however, a single-use model is less established because production-scale single-use TFF cassettes are not readily available. Novasep (through TangenX Technology Corp.) offers the first presanitized, purposely built, single-use TFF cassette for the biopharmaceutical industry.
Sius™ cassettes have been designed to offer performance comparable with that of reusable products at a fraction of the cost. Each cassette arrives presanitized, ready to be equilibrated with buffer and used for processing. Prior to shipment, each is 100% integrity tested and certified for both low TOC and low endotoxin. Sius™-LS cassettes are available in a range of surface areas from 0.01 m
2
to 1 m
2
, and Sius™ process cassettes range from 0.5 m
2
to 5 m
2
. These cassettes have been developed using an innovative manufacturing approach that provides an efficient production process yielding a single-use cassette costing significantly l...
Figure 1.
Finding optimal conditions for a downstream purification process is critical to achieving high productivity and a robust biopharmaceutical manufacturing process at large scale. In addition, the FDA’s new Quality by design initiative may increase demands on process development work because a higher degree of process understanding must be acquired. This understanding/knowledge can be obtained if a larger experimental space is considered and/or more detailed studies are performed.
The introduction of high-throughput techniques for process development partially addresses these challenges. PreDictor™ 96-well filter plates prefilled with GE Healthcare chromatography media allow parallel screening of conditions for binding, wash, or elution. In this study, Predictor plates were used for screening of loading conditions for conalbumin (a protein showing nontraditional behavior) to the cation exchanger Capto™ S. Corresponding column experiments were run to verify that data correlated between formats.
Expe...
Figure 1.
Accurate and rapid IgG titer determination is critical in the selection of cell lines and culture development for antibody production. ForteBio’s Octet QK and Octet RED Systems provide rapid and accurate IgG quantitation in a label-free format that is easy to use. The Octet family of instruments offers analytical capabilities that provide greater value in applications where existing methods such as HPLC and ELISA have limitations in throughput, performance, workflow, and ease of use.
Principle
Different protein concentrations result in different binding curves, which are plotted against their known concentrations to generate a standard curve. The concentration of unknown samples is calculated based on the binding rate from the standard curve.
Key Features of the Octet QK and Octet RED for Antibody quantitation
Key Bioprocess Applications
Figure 1.
CIM Convective interaction Media® are monolithic chromatographic supports used for purification of plasmid DNA, viruses, and other large biomolecules on a laboratory and industrial scale. CIM® monoliths are characterized by large flow-through channels (average diameter of 1,500 nm), which enhance the mass transfer between the mobile and stationary phase. This results in a high and flow-independent dynamic binding capacity and resolution. For plasmid DNA purification on an industrial level, these characteristics increase productivity, making CIM® monolithic columns attractive for industrial applications (
1
).
BIA Separations has developed a CIM®-based pharmaceutical-grade plasmid DNA purification process that can be used on both laboratory and industrial scales. One key step was the development of a new hydrophobic-interaction monolith, CIM® C4 (butyl). This HIC step results in the removal of open circular plasmid DNA from the supercoiled form, thus producing a final plasmid DNA product with >98...
+1 Figure 1.
A virus validation study is required for virus-removal filters prior to employing them in a biopharmaceutical purification process. Validation conditions are determined on a case-by-case basis by each filter user. Optimizing conditions for removal of parvovirus is important to achieve LRV = 4. However, the optimal conditions are often unclear for validation studies, because each product may behave differently. We attempt to establish optimal conditions for human IgG and show the effects of varying IgG concentration, purity of the virus spike, and the spike percentage on the performance of Planova® virus-removal filter in a model validation study.
Materials and Methods
For the porcine parvovirus (PPV) preparations used in the spiking experiments, serum or serum-free PPV supernatants were collected, centrifuged at low speed, and 0.45-µm filtered to obtain stock solutions. In the case of purified PPV, following 0.45-µm filtration, ultracentrifugation, and density gradient centrifugation was applied...
Figure 1.
As a leading provider of process and analytical solutions for the biotechnology and pharmaceutical industries, Applied Biosystems offers an extensive portfolio of tools to help optimize every stage of biopharmaceutical production — from characterization to purification to pharmaceutical manufacturing. These solutions provide pharmaceutical manufacturers with the tools they require to maximize throughput and yield and ensure product quality.
Purification
High Performance on a Large Scale:
Applied Biosystems POROS Perfusion Chromatography products are today’s best-performing chromatographic media for process-scale bioseparations. These rigid, robust particles enable high-resolution separations with two to three times the throughput of conventional fast-flow gels. They are easier to handle and pack, and they offer outstanding cleanability. All POROS process-scale media products are backed by full regulatory support information in a drug Master File. POROS media are used in the manufacture of numer...
Figure 1.
Recently, purification of biomolecules, such as peptides and proteins, by process scale HPLC using reversed phase silica gel media has become increasingly important in biopharmaceutical production. DAISOGEL SP-300-C4-BIO is the newest product using this technology, a butylsilyl(C4)-modified silica gel with 30-nm pore diameter for applications involving peptides, proteins, and other large molecules. Besides selectivity, several other aspects influence the performance of chromatography in preparative processes and consequently have a crucial effect on the total process cost. Therefore, when we developed this product, we focused on properties such as loadability, recovery, mechanical stability, and chemical robustness.
Loadability has been believed to depend on the physical surface area of a packing material, which is typically measured by N
2
adsorption (BET method). Consequently, smaller pores (e.g., larger surface area) should lead to increased loadability. However, this principle is misleading...
Wacker Biotech has a 20-year track record in manufacturing microbial-derived biopharmaceuticals, giving us a deep understanding of such products and of our customers’ needs. We provide comprehensive contract-manufacturing services and proprietary technologies that significantly lower the cost of goods. Our customers benefit from a comprehensive service for the development of robust, highly efficient, and readily scalable processes and GMP-compliant manufacturing for clinical trials and commercial supply.
Service
We work in close collaboration with our customers. Projects are managed by dedicated project managers supported by cross-departmental teams. Depending on our customers’ requirements, we have the flexibility to perform the whole process, or transfer projects at various stages. Wacker Biotech’s services comprise the full range — from construction and optimization of host/vector systems to filling of the bulk API. Construction of production strains, process development in fermentation and downstream ...
Pressure is bearing down on all sides. Pharmaceutical and biotechnology companies face stress from increasing costs, intensifying global competition, growing regulatory stringency, and more. For years, pharmaceutical and biotechnology companies have relieved a little of that pressure by outsourcing functions to a limited extent, such as in packaging or distribution. Allowing a contract manufacturer to perform some of these operations frees up companies to focus on their core competencies.
Today, however, some of the industry’s most eye-opening success stories are the result of even more extensive outsourcing strategies. Effective outsourcing in the biotech and pharmaceutical industries requires more than simply finding a contract manufacturer who can perform restricted tasks. For a truly successful outsourcing strategy, companies must find a true partner.
Contract Manufacturing Basics
Of course, this all starts with effective and reliable manufacturing techniques. By choosing the right contract manufactur...
The biopharmaceutical industry is a predominantly research-based industry that is rapidly maturing toward commercialization. As it does so, biopharma executives have to grapple with complex issues concerning process development, scale-up, and preclinical manufacture of material for toxicology studies, clinical studies, and eventually commercial sale. Selecting a suitable CMO, negotiating a robust contract, and managing on-going relations are of utmost importance to successfully securing a timely, quality supply of product.
There are very different types of CMO. Some CMOs are biopharma companies with their own product(s) under development that rent out surplus manufacturing capacity. Most clients avoid surplus-capacity CMOs because of issues with security of supply. If a surplus-capacity CMO is small, it carries the inherent risk of failure associated with its own clinical products and subsequent corporate instability. On the other hand, if a surplus-capacity CMO is large and successful, then its own in-ho...
Rentschler Biotechnologie GmbH is a full-service contract manufacturer with over 30 years of experience in the development, production, and approval of biopharmaceuticals in compliance with international GMP standards with a highly skilled staff of 350. As part of the Rentschler Group and headquartered in Laupheim, Germany, Rentschler is one of three leading European CMOs operating globally. Dedicated to delivering high-quality biopharmaceuticals produced in mammalian cell culture, Rentschler has nine stand-alone GMP suites with volumes of 30 L, 250 L, 500 L, and 2,500 L, which allows production of material for clinical trials and supplying the market. Rentschler Biotechnologie is a pioneer in the development and production of biopharmaceuticals
—
it was the first company in the world to gain market authorization for an interferon-containing drug.
Rentschler Excels with Expertise and Full Service from “Gene to Drug Product”
Rentschler provides customized, integrated biopharmaceutical services: from cell...
Founded in 2000 in Grenoble, France, Protein’eXpert is an expanding contract research organization devoted to recombinant protein engineering and production. Our 50-member staff offers custom-designed services focusing on the discovery, optimization, and production of valuable target or therapeutic proteins.
Since 2004, Protein’eXpert has extended its offering to the biomanufacturing of therapeutic protein and recombinant vaccines for early clinical trials through its integrated subsidiary PX’Pharma. PX’Pharma provides dedicated contract services for process development and scale-up optimization as well as preclinical and clinical CGMP manufacturing.
Integrated Organization for Seamless Transfer
We differentiate from other protein CMOs by our integrated way of working from early stage research to phase 1 and phase 2 clinical production. This original approach ensures seamless transfers and therefore leads to significant reduction in protein development–related budget and time. Our programs are set up to e...
Trust, competence, and motivation are the major characteristics of a reliable contract manufacturer for clinical projects. At Miltenyi Biotec, customers have more than just projects: they have goals that Miltenyi Biotec helps them to achieve. Miltenyi Biotec is a midsized biotechnology company, focused on medical devices, equipment, and therapeutics for R&D and clinical applications. The product expertise includes GMP-grade antibodies, peptides, growth factors, cytokines, aphaeresis materials, and cellular products, resulting in a broad and significant understanding of different manufacturing challenges. Miltenyi Bioprocess, the contract manufacturing business unit, offers customers comprehensive GMP-compliant contract manufacturing services that encompass the entire life span of their products, from early stage clinical trials to commercial supply.
Manufacturing
Miltenyi Bioprocess manufacturing services for prokaryotic and eukaryotic cells start with cell banking and cover process development, USP, DSP,...
The biopharmaceutical industry is faced with a challenging task: planning, developing, manufacturing, and bringing to market a highly specialized and complex biopharmaceutical product. Competitive pressures, cost, and — above all — time to market are of key concern.
The wide range of implementation options, from in-house resources to outsourcing companies of various calibers and capabilities, adds yet another layer of complexity to the decision-making process. Our objective is to offer our customers a full-service alternative to the development and manufacturing portions of this process. We offer a proven, fully integrated facility staffed with skilled professionals in each critical area. Our management and staff combine years of experience and capabilities in manufacturing both clinical-trial and commercial biopharmaceuticals.
Although some of our capacities and capabilities are comparable with other contract services, it is our unique focus on and experience with products expressed by mammalian cells th...
KBI Biopharma is a leading contract development organization for the biopharmaceutical industry. We accelerate and optimize drug development and manufacturing programs for our client partners, including global pharmaceutical and biotechnology companies and organizations in biodefense. Our clients benefit from faster time to market and lower cost of manufactured goods.
Ongoing Innovation
We are continually working on new technologies and new partnership opportunities to enhance biomanufacturing. Our expertise in biophysical and biochemical protein characterization makes us highly successful in delivering optimal product stability and bioactivity for our clients. Biophysical characterization leads to optimized, predictable processes and rugged, effective formulations that allow for rapid approval, lower costs of goods and a decreased regulatory burden. We excel in formulation and analytical development and have positioned ourselves to expand our capabilities in other areas of biopharmaceutical product devel...
With expansion of production capacity, range of services, and customer base, CMC continues to grow. Earlier this year, CMC acquired the former ICOS Biologics development and manufacturing facility in Seattle, WA, from Eli Lilly. Now known as CMC ICOS Biologics, this facility focuses primarily on mammalian production, and during 2008 it is being upgraded to enable production of in-market biopharmaceuticals as well as late-stage clinical trial material. Dr. Gustavo Mahler, previously with Bayer Healthcare, has been appointed president of CMC ICOS. With this acquisition, CMC has significantly increased its production facilities as well as added additional services including formulation and cell line development. With facilities in both the United States and Europe — and many satisfied customers on both continents — CMC has become one of the world’s largest biopharmaceutical contract manufacturers. With the acquisition of ICOS Biologics, CMC acquired the rights to the CHO cell line expression technology, CHEF...
The covalent attachment of polyethylene glycol (PEG) to a therapeutic protein is frequently used to increase the half-life of that protein in patients while reducing their immunogenic response. The PEGylation reaction can be optimized to maximize the desired product (average moles of PEG per mole of protein) molecules while limiting undesired byproducts (under- and over-PEGylated species). In the example discussed here, the desired molecule was mono-PEGylated, yet the preoptimized reaction yielded multi-PEGylated species visible by SDS-PAGE. The goal of our experiments was to maximize the amount of mono-PEGylated product produced during the PEGylation reaction while minimizing the costs associated with manufacturing the mono-PEGylated product.
Protein X is a novel therapeutic protein produced by the
Pichia pastoris
expression system. After purification, Protein X is reduced and PEGylated, then the mono-PEGylated species purified. We determined the structure of the experiments to optimize the reaction us...
+1 HCM is a leader in upscale process development and aseptic filling of liquid parenterals in syringes, vials and custom containers. HCM offers clients–from newly-established firms to large-scale commercial enterprises–high quality services and innovative technologies. Quality Services
Quality Services
Foremost among the services HCM offers are upscale process development and aseptic filling of syringes, vials, and custom containers. In addition to traditional fill and finish, HCM offers online vacuum filling and stoppering as well as lyophilization. HCM offers customers a Stability optimization Service (SOS) to assist them in determining the optimal container presentation for their products. HCM’s areas of specialization include difficult-to-process viscous and suspension products, microfluidization, homogenization, low-shear mixing, lyophilization, and aseptic diafiltration.
HCM’s Burlington, MA facility houses 60,000 ft
2
of manufacturing, laboratory, cleanroom, and office space and employs 125 highly t...
Eden Biodesign is one of Europe’s leading contract biomanufacturers. We are an expert provider of consultancy, biopharmaceutical process development, and CGMP manufacturing services. Our rarely found blend of experience and expertise, gained from providing our services to more than 100 clients worldwide, ensures that the clinical medicines we develop are regulatory compliant and optimally positioned for further clinical and commercial development. Our state-of-the-art facility is licensed to produce clinical products, which we believe is best in class for its scale of manufacture, whether for microbial or mammalian recombinant proteins, cell products, viral vaccines, or gene therapies.
Our primary goal is to be open and honest. We all know that the path to biopharmaceutical glory is not always smooth, but the truth is that we will never seek to hide anything from you. We will keep you informed of setbacks as well as breakthroughs. We don’t develop proprietary products, and although we provide access to th...
The Boehringer Ingelheim group is one of the world’s 20 leading pharmaceutical companies. Headquartered in Ingelheim, Germany, it operates globally with 135 affiliates in 47 countries and nearly 39,800 employees. Since its foundation in 1885, the family-owned company has been committed to researching, developing, manufacturing, and marketing innovative products of high therapeutic value for human and veterinary medicine. Our therapeutic areas are respiratory, cardiovascular, central nervous system, urology, virology, rheumatology, metabolism, immunology, and oncology.
Boehringer ingelheim is one of the leading companies for industrial customer manufacturing of biopharmaceuticals by offering the entire production technology chain in development and production at its biopharmaceutical facilities in Biberach (Germany) and in vienna (Austria).
The large-scale manufacturing sites deliver biopharmaceutical products like therapeutic proteins, fusion proteins, protein scaffolds, monoclonal antibodies, antibody mi...
Catalent Pharma Solutions offers a total solution for the development and manufacture of biopharmaceutical and sterile products. Our proprietary technology — and expertise in working with proteins, peptides and other biotechnology-derived active materials and small molecules — helps to maximize the potential of your API and accelerate your product’s speed to market.
From cell line engineering to manufacture of API and Drug Product for clinical trials, our team of highly experienced protein chemists, biochemists, pharmacists and pharmaceutical scientists provide the exceptional service, focus and expertise that your project needs.
Setting a New Standard in Mammalian Cell Line Engineering and CGMP Manufacturing
Catalent’s proprietary GPex® (Gene Product Expression) technology enables genetic engineering of highly stable, high-yielding mammalian cell lines with unmatched speed and efficiency. It has been used around the world to produce material for clinical trials and offers many important benefits includin...
