Cation exchange resins are used in many protein purifications. The high capacity of these resins is very important to process throughput particularly as protein expression levels reach titers >10 g/L. In many cases the feedstock may need to be diluted before loading onto a cation exchange resin to maintain the high protein dynamic binding capacity (DBC) reported by the manufacturer. The additional water, buffer volumes, and process time add cost to the loading step.
Tosoh Bioscience is pleased to announce the new TOYOPEARL
®
MX-Trp-650M resin, which shows both high DBC and tolerance to feedstocks with conductivities up to 17 mS/cm. The new mixed-mode (MM) resin has also been engineered to have excellent elution kinetics.
The data show that TOYOPEARL MX-Trp-650M resin
TOYOPEARL MX-Trp-650M is the optimal resin for protein capture steps where the selectivity and protein clearance needed is lower than with affinity resins. This novel resin an excellent choice for intermediate purifications.
Resin Structure
Single-use technologies are a growing market and becoming increasingly common in biopharmaceutical manufacturing. Because of this growth and their increasing use in further reaches of upstream and downstream processes, understanding the safety and sanitation of single-use technology is vital.
One of the key benefits of single-use systems is their potential to eliminate cross-contamination during pharmaceutical manufacturing. In processes that are entirely single-use, replacement of the fluid path assembly during production eliminates the concern for contamination in any valve or line.
Safe from the Start
A quality, sterile product comes from a quality, sterile manufacturing partner who understands the critical nature in which single-use systems will be used. ASI is an ISO 13485:2003, FDA-registered manufacturer that has built upon regulatory foundations through the use of a customized quality management system intertwined with enterprise resource planning (ERP) software. From incoming raw material inspect...
The utility of insect cells for protein production has been described in several reviews. Grown in batch mode, production cell lines Sf-9, Sf-21, and BTI-TN-5B1-4 can reach high cell densities (>1 × 10
7
cells/mL) in an optimized culture medium. Alow-shear cultivation system that guarantees sufficient oxygen supply is also required. The
k
L
a
parameter is used to characterize oxygen transfer in a cultivation system.
The 250-mL shaken flasks used in this study were coupled to a Shake Flask Reader (SFR) provided by PreSens. The SFR unit has nine modules enabling wireless real-time monitoring of pH and DO. Using the standard gassing-out method, nine experiments at varying fill volumes (25–150 mL) and shaking frequencies (80–200 rpm) were performed. DO concentrations were monitored over time, and experimental plus model-derived
k
L
a
values were calculated (
1
). Experimental
k
L
a
values ranged 4.4–37.9 h
−1
, corresponding to the modeled oxygen transfer data (data not shown).
Propagations of Sf...
Figure 1: ()
The production of therapeutic proteins like monoclonal antibodies (MAbs) and bispecifics depends on mammalian expression due to the posttranslational modifications occurring in these molecules. At Boehringer-Ingelheims (BIs) BioXcellence unit, we use the BI-HEX™ mammalian expression platform for fast and cost-efficient development of high-titered manufacturing processes. The BI-HEX™ platform is composed of the following interlinked elements: BI’s proprietary vector system, the BI-HEX™ cell lines, which are derived from the CHO-DG44 cell line (
1
), a short and efficient manufacturing process, as well as BI proprietary media and feeds. Use of automation and robotic systems as well as miniaturization of many of the processes used during cell line, upstream, and downstream development are key features of the platform. For example, the ambr system was recently introduced for downscaled fed-batch clone screening.
The BI-HEX™ platform is versatile in the sense that it is made of variations of each ...
+7EMD Millipore has developed a family of single-use bioprocess containers designed for mammalian cell growth and recombinant protein production. The Mobius
®
CellReady product offering includes bench-scale (3-L), small-scale (50-L) and pilot-scale (200-L) bioreactors spanning early process development through clinical batch production. To enable the successful scale-up of a biomanufacturing process, a number of factors critical to efficient cell growth, viability, and protein production were considered and used to develop an optimized design. These include mixing efficiency, gas transfer capability, and an operating range of power input that minimizes cell shear. In this study, several key design parameters such as oxygen mass transfer coefficient (
k
L
a
), power per unit volume, Reynolds number (Re), mixing time, and tip speed were characterized for the three different sized single-use bioreactor containers. Based on these data, CHO cells were cultured in each of these bioreactors by maintaining equival...
+3 Packed-bed basket technology developed by New Brunswick Scientific (acquired by Eppendorf Inc. in 2007) provides a shear-free environment for production of animal cells. Because little information is available on the utility of the New Brunswick CelliGen BLU single-use bioreactor system for production of secreted proteins (especially in perfusion mode), this study was conducted to measure growth and productivity of alkaline phosphatase (ALKP)–secreting rCHO cells. We used two packed-bed bioreactor types — a 5-L New Brunswick CelliGen BLU single-use vessel and a 2.5-L autoclavable glass vessel — both operated by New Brunswick CelliGen 310 console in perfusion mode. Perfusion provides a homeostatic environment for optimal cell growth similar to that experienced by cells in vivo, where waste products are constantly removed and fresh nutrients are replenished. Cells cultured in packed-bed bioreactors are not exposed to hydrodynamic forces, which allows for maximum cell growth and protein expression (
1
). Our...
+1 In order to reduce scale-up costs and facility capital expenses, biotechnology companies are adopting single-use systems. To fully enable the single-use paradigm, automation software, hardware, and single-use sensors must be optimized; moreover, both validation documents and materials certifications must be readily available for cGMP implementations.
Finesse provides turn-key, configure-to-order measurement and automation solutions for many single-use bioreactor types: “stirred-tank,” “paddle,” or “rocker” systems. The Finesse turn-key system is optimized for each bioreactor type to maximize product yields. Finesse systems are scalable from R&D to process development to cGMP manufacturing. Finesse also provides full support for its systems, including installation, training, validation packages (FAT, SAT, IQ/OQ), and service/maintenance programs.
The primary enablers of single-use systems, however, are the sensors. Finesse offers disposable sensors for dissolved oxygen (TruFluor DO), pH (TruFluor pH), temp...
One of the major bottlenecks in the production of biopharmaceuticals is the efficient expression of therapeutic proteins in microbial or mammalian cells. The
Escherichia coli
pAVEway™ expression system described here has been developed to ensure high product titers and efficient scale up to GMP manufacture while minimizing many common issues seen in other expression systems, such as “leaky” expression (expression of recombinant protein in the absence of inducer).
How It Works
The use of a number of powerful
E. coli
RNA polymerase promoters — such as T7A3, λpL, and
tac
— opens up a large host range in comparison with the popular T7 system that is limited to hosts carrying the λDE3 prophage. Control over basal expression is provided by the use of perfectly palindromic
lac
operator sequences (Figure 1). This high level of control is extremely important in large-scale (≥5 L) fermentations because it allows high biomass accumulation prior to induction which can be helpful when expressing proteins that ...
Figure 1: ()
Continously new bioprocesses are evolving, and bioprocessors are searching for the easiest way to process samples in R&D. Currently, a huge trend toward disposable production systems is notable. For these purposes plastic bags, stirred tank reactors, and storage tanks established on the market. Unfortunately, these reactor types are not suitable to perform sophisticated bioprocess development. In bioprocess R&D, throughputs of 50 to 1,000 fermentations are needed. Therefore, m2p-labs developed intelligent new tools at microscale to fulfill the needs in bioprocess development. The combination of high-throughput experimentation, online monitoring, scalability, and automation made it possible to provide unique and very efficient bioprocessing tools at microscale. Our mission is to bring bioprocesses as fast as possible from microreactor to process. m2p-labs tools include the following technologies.
Figure 1: ()
BioLector
®
Technology
The BioLector is a unique high-throughput fermentation system...
We have previously reported rapid, consistent, and highly productive fed-batch processes from shake flasks to 5-L glass and 200-L single-use bioreactors using the ActiCHO Media System (
1
). This system has demonstrated to be highly reliable in many processes using different Chinese hamster ovary (CHO) cell clones. PAA Laboratories/GE Healthcare manufactures this medium in both liquid and powder formats and supplies it worldwide for large-scale applications. Here we present some data on industrial scale-up studies going directly from small shake flasks to 1,000-L bioreactors.
Features of ActiCHO Media System
ActiCHO Media System is chemically defined and therefore entirely free of animal-component ingredients. The system does not contain growth factors, proteins, peptides, or hydrolysates. Most important, it was designed to be a ready-for-use platform and to require no optimization with CHO DG44 cells expressing heterologous genes. This was achieved by formulating a highly balanced set of ingredients that...
Microbial contamination of cell cultures is a major process risk that leads to lost production batches with considerable economic impact for a biopharmaceutical manufacturer. Incidences of penetration of typical 0.2-µm sterilizing-grade membranes by mycoplasma species have led to higher-retention 0.1-µm–rated filters becoming commonplace for filtering mammalian cell culture media.
Increasing Retention without Slowing Down Processing
There are a number of 0.1-µm–rated filters available, and although many are effective at removing mycoplasma contamination, the tighter filtration membrane required is detrimental to the system flow rate. With strict time limits from the addition of the first media component to the end of filtration at media suppliers and biopharmaceutical manufacturing facilities, this can significantly increase the risk of contamination of unfiltered media.
Parker domnick hunter’s new PROPOR MR has been specifically developed using our customers’ feed streams to deliver industry leading flow...
Figure 1: ()
The
Pf
ēnex Expression Technology™ platform is a very powerful protein expression technology based on the microorganism,
Pseudomonas fluorescens
. The Pfenex platform can deliver a robust production strain with unprecedented speed and a success rate that transcends legacy production hosts. Traditional recombinant protein expression hosts such as
E. coli,
insect cells, and yeast tend to have high failure rates regarding their ability to produce high titers of soluble, full-length, active target protein. In addition, the time required to identify the optimal expression strain in these systems is protracted due to a linear and iterative approach to strain engineering, resulting in significant opportunity cost for the drug developer.
Pf
ēnex Expression Technology™ dramatically reduces strain development time and hence product development time through robotically enabled high-throughput, parallel expression strain screening. The resultant selected production strain delivers a lower overall co...
SciLog’s MabTec™ is an add-on bioreactor maintenance system that can gravimetrically manage, automate, and document your bioreactor feeding or perfusion strategy. The MabTec™ upgrades many manual processes to walk-away automation with minimal investment in terms of capital or time.
Summary
MabTec™ combines accuracy with convenience to provide an ideal solution for cell culture feeding strategies. Based on the results, MabTec™ demonstrated the ability to increase protein production while eliminating several hours of manual daily operation.
Figure 1: ()
Introduction
The application flexibility of MabTec™ delivers superior growth efficiency within a bioreactor through the replacement of repetitive manual operator steps with an automated solution. The replacement allows for more constructive use of operator time and eliminates the inadvertent errors associated with manual production. The objective of the experiment is to
Test Scenario
Three ten liter (10 L) glass bioreactors were filled with four liters (4 L)...
NNE Pharmaplan has developed a new facility concept we call Bio on demand™. Facilities based on the Bio on demand™ concept can be established in one to two years. The result is a flexible facility that is fully operational, locally compliant, and with functioning quality systems. The Bio on demand™ concept involves a high degree of single-use technology to ensure cost-effective production and fast establishment.
Facilities based on our Bio on demand™ concept can be delivered globally, but they are primarily intended for growth markets where time-to-market is particularly essential. We are currently applying the standard Bio on demand™ concept in the design of a number of new biotech facilities for both local and international companies established in emerging markets.
Full Package for Full Predictability
The Bio on demand™ concept includes engineering and supply of a facility as well as related quality systems, standard operating procedures (SOPs) and coordination of necessary quality tests. By not only p...
As the pharmaceutical industry focuses on developing biological drugs derived using cell culture processes, the ability to optimize the growth, health, and potential yield of the cell line becomes increasingly important. Finding the optimal cell culture media is a daunting and time-consuming process due to the amount of potential media components and the unique requirements of the cell line being researched. Osmolality information is a crucial component to this research because of its close relationship between cells and media.
Background
Most current osmometers use freezing point depression technology to measure the total solute concentration of a liquid solution. Freezing point is considered the gold standard method in the pharmaceutical industry due to its accuracy and its broad applicability for almost any liquid sample type including complex media mixtures. Historically, high-throughput osmolality determinations have been limited due to the ability of the systems to process only one sample at a time ...
Rapid microbial testing methods continue to gain acceptance in pharmaceutical manufacturing, but experience shows that most companies still use traditional methods for their testing. The ability to deliver results in less time than traditional methods provides compelling benefits to manufacturers. Two key areas of microbial testing where these benefits can be realized include environmental monitoring and sterility testing.
Microbial testing must be completed throughout the manufacture of pharmaceuticals, including the raw materials phase, in-process phase, and release-testing phase. The use of rapid methods during these steps can reduce time and error; however, most companies still use methods that were popular in the 19th century.
Wider acceptance of rapid methods is hampered by constraints such as the method’s focus on one or two applications. Some methods are suited for water and bioburden, others for air, and others sterility. Some have more than one application, but not all. Some methods use reagents...
