Product Lifecycle Management

Jayne Hesley, applications scientist in marketing for Molecular Devices, Inc. (Sunnyvale, CA)

Homogeneous Antibody Binding Assay: Comparing Imaging Systems and Optimizing Acquisition Parameters for High-Throughput Screening

Hybridoma cell lines are cultured to produce monoclonal antibodies for use in diagnostics, vaccine development, or therapeutics. One well-accepted assay for screening the antibody-containing supernatant of hybridomas is to capture the antibody on the surface of beads (<10-μm diameter microspheres) and then measure the captured amount by tagging with a fluorescently labeled secondary antibody and imaging the beads' fluorescence. A similar assay can be done to measure ligand-binding on cell surfaces. Robust homogeneous assays are straightforward to develop and these assays can be automated and scaled up to 1,536-well plates because no washing away of excess antibody or label is required. These studies show that the IsoCyte DL scanning cytometer achieves sensitive and reproducible detection of both cell-and bead-surface fluorescence while rejecting fluorescent background from the solution in the well. In addition, a plate of any well density can be scanned and analyzed on-the-fly in 5–10 minutes. Optimization of assay and acquisition conditions such as fluorophore wavelength and scan settings are presented as well as a comparison between the traditional ABI8200 FMAT platform, IsoCyte scanning cytometer, and ImageXpress Ultra confocal imaging system.

Take-Home: A homogeneous antibody-binding assay is suitable for screening hybridoma supernatants in research, production, or quality control environments. This assay is straight-forward to optimize and to scale into a 384-or 1,536-well microplate format.

Cell Culture and Upstream Processing

Melisa Carpio, research associate II in process sciences at Takeda San Francisco (San Francisco, CA)

Evaluation and Implementation of a 24-Well Deep-Well Plate System for a High-Throughput Cell Line Selection Platform

Creating a production cell line for clinical manufacturing is a time-intensive process that typically involves funneling a large number of clones through various screening steps before finally selecting a lead. Incorporation of a high-throughput fed-batch culture system would greatly facilitate this process by eliminating the need for any intermediate screening steps and allowing a larger number of candidate cell lines to be evaluated, thereby increasing the probability of success. Therefore, we evaluated two different culture systems — spin tubes (ST) and 24-well deep well plates (24DWP) — as higher-throughput alternatives to traditional fed-batch shake flasks. For nine cell lines evaluated, the ST and 24DWP both exhibited strong correlation to the shake flask with regard to antibody titer (R² = 0.93 and R² = 0.94, respectively). This translated to nearly identical relative clone rankings in the case of the 24DWP and shake flask (R² = 0.93). The viable cell density correlation was also strong through the growth phase of culture (R² = 0.74 for ST and R² = 0.83 for 24DWP) but decreased at later points, mainly due to the formation of cell clumps. Finally, rates of evaporation were similar between the various devices over the time duration studied. Based on the significant advantages the 24DWP represents in both ease of sampling and throughput (sixfold increase compared to shake flasks), they have been incorporated into the TSF cell line development platform. Application of this approach to generate cell lines for a Takeda antibody yielded high-expressing clones (> 2 g/L) in about two months post transfection. Final selection of stable lead cell lines was possible in under five months, representing a time-savings of one to two months over previous approaches.

Take Home: We have developed a high-throughput 24-well deep well plate system that has a strong cell growth and antibody titer correlation to the shake flask. Implementation of this system into our cell line development process has allowed us to select stable cell lines producing >2 g/L in under five months.

Sally Grosvenor, scientific communications manager in application research and development at Novozymes Ltd. (Wellington Point, Queensland, Australia)

Adaptation of CHO Cells to a Growth Factor Supplement Improved Growth Rate and Productivity

Prior adaptation of Chinese hamster ovary (CHO) cells to their culture environment is critical for optimal performance in upstream manufacturing processes. The importance of adapting cells has been previously demonstrated with the evolution from culture media containing serum to a serum-free and/or chemically defined environment. However, although these cell lines are optimized to their culture surroundings, process development scientists often discover suboptimal performance compared with serum-containing media. To address that, supplements such as growth factors are often used. This study demonstrates the requirement for adapting CHO cells to the LongR3IGF-I growth factor supplement for improved growth rate and productivity. Briefly, CHO cells were adapted from an undefined serum-containing media to either a commercially available chemically defined media (CDM) or a combination of CDM and LongR3IGF-I supplement over a number of passages until a stable doubling time was observed. Master and working cell banks were generated from cells derived from this adaptation process. Cells from both working cell banks were then tested for growth and productivity performance in either CDM alone or in combination with LongR3IGF-I growth factor.

Take Home: Adaptation of CHO cells to production media is crucial for optimal cell perfromance.

Rachel Legmann, principal investigator in applications at Seahorse Bioscience (Billerica, MA)

Accelerating Development and Ensuring Product Quality of Biosimilars Using a MicroBioreactor Platform

The strategy to rapidly define process and quality criteria for biologic therapies is the use of multifactorial, statistically designed experiments (DoE). Environmental and metabolic factors during a bioprocess can affect the efficacy and safety of the glycoprotein. The automated SimCell platform can run hundreds of experiments in parallel using microbioreactor cards designed to simulate a bioreactor environment by controlling pH, dissolved oxygen, and glucose. The throughput of this system challenges many conventional downstream analytical techniques that can’t handle small harvest sample volumes and higher throughput. We addressed this problem by using a lectin-array based glycoanalysis platform for quantitative glycan profile assessment. Four clones were screened in four different media formulations, under several process variations to determine what condition give the correct conditions to produce a titer and glycosylation profile similar to that of a reference drug. A total of 240 microbioreactors were processed, followed by immediate downstream glycan analysis. The best and worst combinations were obtained with the same clone but different media. This study demonstrates clearly that using m
ultidimensional screening for clones and process parameters enables targeting of a specific glycosylation profile to be as biosimilar to the reference drug without compromising titer yield.

Take Home: Added value and synergistic effects can be obtained with miniaturized, controlled bioreactor systems in combination with fast and easy glycoanalysis capabilities for high-throughput screening of clones and process conditions.

Jim Blasberg, senior R&D scientist in analytical R&D at Sigma-Aldrich (St. Louis, MO)

Development of Analytical Methods for Identifying Bioactive Components in Protein Hydrolysates

Protein hydrolysates are complex materials commonly used in biopharmaceutical cell culture processes. Hydrolysates are known to have a substantial positive impact on cell growth and/or protein production. Given their undefined and variable nature, a need exists to develop a chemically defined alternative that provides similar performance. Toward this goal, reversed-phase HPLC fractionation followed by cell culture screening was used to isolate “bioactive” fractions. This presentation will highlight the evolution of chromatographic and mass spectrometric methods developed for identification of bioactive components from complex mixtures that led to the creation of a chemically defined alternative to protein hydrolysates.

Take Home: Protein hydrolysates are very complex. Analytical techniques are valuable in identifying bioactive components of these complex mixtures.

Nicolas Pierre, scientific consultant in sales and marketing at Cisbio US (Bedford, MA)

Investigation of Tag-lite Applications for Screening Biotherapeutics on Cell Surface Protein Targets

Through this poster we show how Tag-lite technology, a new assay principle, can be used to screen therapeutic antibodies for their binding to cell-surface protein targets and characterize their affinities. Different assay formats were applied to determine the binding of antibodies to two different receptors: CXCR4 (a GPCR belonging to the chemokine family) and EGFR (a tyrosine kinase receptor). Binding of the antibodies to their targets was detected using Tag-lite, a combination of HTRF and SNAP-tag technologies. Specific cell lines were designed for each receptor allowing either binding experiments or functional assays (cAMP, Phospho-Erk) as well as internalization tests.

Take Home: In this study, we demonstrate that Tag-lite technology can detect antibody binding on cell surface receptors. The technology offers a lot of flexibility in terms of assay format, thereby enabling the detection of different binder types, the determination of their binding affinity, as well as their ranking. Being HTS friendly and highly miniaturizable, Tag-lite shows obvious advantages for cost-effective screening of therapeutic antibodies.

Hartmut Tintrup, director of marketing and business development for Cevec Pharmaceuticals GmbH (Cologne, Germany)

CAP-T Cell Expression System: A Novel Rapid and Versatile Human Cell Expression System for Fast and High-Yield Transient Protein Expression

Human CEVEC’s amniocyte production (CAP) cells allow for stable, high-level production of recombinant proteins with excellent biologic activity and therapeutic efficacy as a result of authentic posttranslational modification. Based on human CAP cells, the new transient CAP-T system has been developed to enable extremely high production yields of even very complex proteins within a few days.

Michael Cunningham, senior research scientist in the bioprocess division of Millipore Corporation (Bedford, MA)

A Case Study Describing the Characterization of a 3-L Single-Use Bioreactor as a Replacement for Glass Stirred-Tank Reactors

The need for faster cycle times and higher levels of operational flexibility has accelerated the adoption of single-use processing solutions in the biopharmaceutical industry. Millipore recently introduced the Mobius CellReady 3-L bioreactor, a single-use vessel that shares all the important features of conventional glass bioreactors used to optimize mammalian cell growth and expression. This single-use, stirred-tank bioreactor is gamma-irradiated and provides for maximum operational flexibility with prefitted weldable tubing, gas inlet filters, a vent filter, and two commonly used sparging options. In this case study, the functional comparability between single-use and glass bioreactor vessels will be presented. Important performance indicators such as CHO cell growth and expression, vessel mixing times, temperature mapping, and volumetric mass transfer coefficients (k_La) for oxygen will be discussed, along with the impact on cell culture performance of using a modified pitched blade impeller in the single-use bioreactor. Using Applikon ez-Controller systems, both bioreactor systems achieve similar k_La values, temperature mapping, and mixing times with only minor differences in operating set points. Despite minor operational differences, both bioreactor vessels exhibit equivalent cell culture performance. The impact of reduced cell culture cycle times on biotherapeutic process capabilities and capacity will also be discussed. In this case study, we will present results demonstrating that the Mobius CellReady 3-L disposable bioreactor vessel is an effective and convenient substitute for traditional glass stirred-tank bioreactors in process development laboratory activities.

Take Home: Single-use bioreactor technology increases process development efficiency and capabilities. Single-use biorectors are designed to mimic conventional glass stirred-tank reactors and in-process data shows them to generate comparable performance.

Yasuhiro Takenaka, new products development in aminoscience at Ajinomoto USA (Kawasaki, Japan)

Effective Protein Secretion Platform Using Corynebacterium glutamicium

Once previously used for amino acid production, the bacterium Corynebacterium glutamicum has been found to have applications within novel protein secretion systems. Complex human proteins for pharmaceutical use as well as industrial enzymes have been efficiently expressed and secreted into culture medium with the correct conformation and high activity, enabling downstream processes to be simplified due to its high purity and productivity. The high–cell-density fermentation process has been scaled to higher volumes, making possible the manufacture of commercially valuable proteins for pharmaceutical use without cell disruption, refolding, or complex chromatographies.

Take Home: Novel protein secretion systems to express complex human proteins for pharmaceutical use or for industrial applications can be achieved through the use of Corynebacterium glutamicum. The expression process using Corynebacterium glutamicum overcomes traditional microbial expression difficulties by secreting correctly folded, active proteins directly into the growth media, greatly reducing costly and time-consuming downstream purification steps, lowering production costs, and shortening overall manufacturing time.

Philip Beckett, senior scientist in applications at GE Healthcare (Piscataway, NJ)

Increased Process Understanding Through Characterization of Host-cell Protein Patterns

Many pharmaceuticals such as monoclonal antibodies are produced from genetically engineered mammalian cell lines. Purity with regard to undesired host-cell proteins (HCP) of final product is essential for product safety. Process development by varying upstream conditions and analyze the effects on downstream processing is needed for optimal yield and purity. We have characterized HCP patterns in purification of monoclonal antibodies from CHO cells by using 2-D fluorescence difference gel electrophoresis (2-D DIGE). Methods 2-D DIGE technology with two different sampl
es and a pooled internal standard per gel prelabeled with CyDye DIGE Fluor minimal dyes, detects differences in protein abundance. Experimental variation is virtually eliminated and the quantitative data is very reliable. Samples from culture supernatant or affinity eluent fractions were buffer exchanged and concentrated into DIGE labeling buffer using membrane filtration columns. A set of three spiked proteins were added to all samples including the pooled internal standard before CyDye labeling. Protein samples and pooled internal standard were labeled with CyDye minimal DIGE fluors and subjected to 2-D electrophoresis. Differences in protein levels between samples were analyzed taking advantage of the spike proteins for sample-to-sample normalizaton and specialized analysis software

Take Home: Differences in protein expression between culture supernatants grown with a set of altered media compositions were analyzed. Differences in the HCP patterns of affinity eluent fractions were analyzed. The results were related to yield of target protein and HCP levels obtained with ELISA assay.

Allison Tanner, senior development engineer in product development at Corning Life Sceinces (Kennebunk, ME)

Development of Corning HYPERStack Multilayer Cell Culture Vessels

The HYPERStack vessels have been designed and constructed to be closed cell culture systems for use in the cell therapy and vaccine production markets. They are the second series of products in the Corning Life Sciences gas permeable family of products. The first product in this family (HYPERFlask) was introduced in 2008 as a product for the high-throughput screening industry. Extensive analysis of oxygen concentrations in media during cell growth in traditional cell culture vessels with headspace was conducted. This included analysis of oxygen concentrations in media during cell growth, oxygen gradient formation through the media based on proximity to the cell layer, and limitations to diffusion by traditional vent caps. Selection of gas permeable film thickness and tracheal space requirements of the gas permeable cell culture vessels is also examined, as well as the principles of operation of gas permeable cell culture vessels.

Take Home: Corning introduces new HYPERStack multilayer cell culture vessel for cell therapy and vaccine production markets. Technical analysis indicates improved environment for cell growth. Cellular gas exchange occurs through gas permeable film. Gas permeable technology allows to improve on limitations of vented cap vessels.

Vitaly Klimovich, senior cells applications scientist for Corning Life Sceinces (Kennebunk, ME)

HYPERStack: Space Saving Scale-Up Solution for Bioprocessing

Corning introduces the new line of modular cell culture vessels featuring gas permeable technology to provide scale up solutions for cell therapy, vaccine production, and protein expression. The vessels feature closed-system accessories for bioproduction application. This novel approach allows achieving space savings by growing more cells per volumetric footprint. Several commonly used cell lines were analyzed for cell yield and viability. Crystal violet staining was conducted to verify uniform cell distribution within the vessel and among the layers. Extensive analysis of cell growth environment such as dissolved gasses, electrolytes, amount of nutrients, and cellular metabolites was done. Analysis of both CO₂-dependent and CO₂-independent approaches were considered.

Take Home: Corning introduces the new HYPERStack line of modular cell culture vessels featuring gas-permeable technology. Multiple cell lines were analyzed for yield and viability. Cell-growth environment was extensively analyzed.

Michael Biksacky, president of enginneering for Flownamics Analytical Instruments, Inc. (Madison, WI)

Application of An Automated Sampling System for Cell Culture Process Development

It is well known that glucose plays an important role in mammalian cell culture as carbon and energy sources. Therefore, it is important to understand the metabolism of glucose to develop strategies to maximize recombinant protein production and control product quality. In the present study, experiment was designed to evaluate the impact of programmed feeding on culture performance by an automated sampling system interfaced with a biochemistry analyzer interfaced with bioreactors. The automatic feed control resulted in smaller change intervals of feed medium as compared to daily manual bolus feeding. Our results showed that the application of an automated sampling system could potentially reduce labor, improve bioreactor efficiencies. In combination with optimized cell culture media and feeds, continuous feeding control could result in condition at which higher total protein production could be achieved, leading to more efficient protein manufacturing as compared to that with generic fermentation process. Product quality was examined and reverse phase HPLC data are presented.

Take Home: Cell culture fed with an automated sampling system can show lower lactate and osmolality levels, which could be beneficial to cell growth and protein production in CHO system. Enhanced productivity (e.g., 15–20% higher) was observed in the current study controlled and fed by an automated sampling system.

Carrie Weymer, associate scientist II in process cell culture at MedImmune (Gaithersburg, MD)

High-Titer/High-Throughput Cell Culture Process Development

As the number of candidate biotherapeutic drugs increases, the need for high titer yielding, efficient production processes to supply the clinic and maximize clinical manufacturing capacity has become apparent. A fed-batch process is routinely used throughout the industry because of its ability to deliver high product concentration and its operational simplicity. Common fed-batch development approaches focus on maximizing specific productivity (q_p) and/or integral of viable cells (IVC), although it is not atypical for a decrease in qp to accompany an increase in IVC. MedImmune’s CHO cell culture platform feeds were enriched, and reformulated in order to achieve an improved balance of the nutrients and optimization of the overall feed volume. The current study with a representative cell line focused on feed development optimization and process set-points to increase volumetric productivity and optimize process throughput. Additionally, modulation of the viable cell density on day of inoculation was used in order to increase the daily volumetric productivity to >600 mg/L/day, with a peak daily volumetric productivity of >1,000 mg/L/day. These approaches increased antibody productivity by increasing the qp by approximately twofold while still maintaining high IVC. This resulted in an increase in volumetric productivity from approximately 4 g/L to 8 g/L while shortening the duration of the process by four days.

Take Home: As the number of clinical trial programs increase, it becomes increasingly critical to be able to produce sufficient quantities of recombinant protein quickly. To that end, we have developed a scheme to increase process throughput (defined as mg/L/day) by shifting growth and low titer time into the seed tank instead of the bottlenecked production bioreactor.

Recovery and Purification

Jeffrey Bettencourt, senior purification development scientist II for Xoma (US) LLC (Berkeley, CA)

Early Downstream Process Optimization for Definition of Viral Safety is Critical in Platform Process Development for MAbs

Xoma is developing triple antibody cocktails to counter poisoning associated with botulinum serotypes A, B, and E, using platform cell culture and purification processes to accelerate development. Removal of viral impurities and contaminants during downstream purification is critical. Each antibody is adapted to the Xoma platform with a
djustments to parameters (conductivity, pH, loading) to accommodate variations in isoelectric point, relative hydrophobicity and charge distribution. To minimize development time and ensure viral safety before scale-up, Xoma implements process characterization studies for each antibody during early bench scale development at the protein A, Q-Sepharose, and HIC steps using DOE techniques.

Supplementary XMuLV viral clearance tests of the Q-Sepharose and HIC steps at their set points, flanked by two tests at “extreme” combinations of parameters, ensure viral safety. We present characterization and preliminary viral safety data for one antibody: XB10. XMuLV was cleared to below detection at Q-Sepharose set points and at more alkaline “extreme” conditions. Clearance was <4 logs at more acidic “extreme” conditions. HIC delivered comparable 4-log clearance under “set-point” and less retentive “extreme” conditions. Virus was cleared to below detection at more retentive “extreme” conditions, although yield was adversely impacted. Our approach couples platform process development with preliminary evaluation of viral safety, resulting in a robust process that can meet the product quality and viral safety requirements of manufacturing. It also defines an “operational space” for each process step prior to GLP/GMP scale-up and provides a basis for informed definition of operating ranges around set points.

Take Home: Xoma’s platform purification process minimizes development times for antibody drug products. Our strategy of incorporating process characterization studies and viral clearance testing into early process development effectively adapts the unique properties of each antibody to the platform, and demonstrates robustness and viral safety before scale-up and transfer to manufacturing.

Alla Zilberman, director of applications for Semba Biosciences (Madison, WI)

Continuous Protein Purification Using Simulated Moving Bed: Taking the Chromatography Platform to the Next Level

Chromatography as a bioprocessing platform has not kept pace with the demand for lower cost, higher flexibility, and higher productivity in the manufacture of protein-based pharmaceuticals. Simulated moving bed chromatography (SMBC) elevates the chromatographic platform by conversion of the conventional batch process to a continuous process. SMBC has been successfully applied to small and large-scale binary separations of hydrocarbons, sugars, and enantiomers but has rarely been used for protein purifications. The highly efficient zones of separation created by SMBC enable continuous bind–wash–elute chromatography and peak shaving of high purity target protein. A bench-scale SMBC instrument was employed to investigate several purification methods including protein A, protein G, immobilized metal affinity, and size-exclusion chromatography.

Take Home: The results demonstrate that the dramatic increases in productivity and purity SMBC offers for small-molecule separations can also be realized for recombinant proteins and monoclonal antibodies. These advantages can help alleviate the bioprocessing bottleneck created by increased protein production upstream making SMBC an improved process alternative to single-column methods.

Mira Linhart, senior research associate in technical operations for Abbott Bioresearch Center (Worcester, MA)

The Effect of Conductivity on Dynamic Binding Capacity on Cation-Exchange Resins

In an effort to improve the antibody manufacturing platform, new cation-exchange resins were evaluated. Dynamic binding studies were conducted using two different MAbs at a stable pH and varying conductivities across nine resins to determine the differences in binding capacity. DBC studies were performed at pH 5 and conductivities of 5, 10, and 15 mS/cm for the nine cation-exchange resins, with breakthrough criteria set at 10%. Although most of the resins provided excellent binding capacity (>50 g/L), the impact of conductivity on DBC was shown to be very molecule dependent and varied significantly with the resin type.

Take Home: A case study is shown where the conductivity dependence behavior for CEX resin DBC is signifcanly different between two molecules.

Lisa Thurston, technical director in business development for PhyNexus (Lowell, MA)

Optimization of Protein Purification Using Small-Scale Separation Columns

Characterization of therapeutic candidates requires that proteins are well purified and enriched post expression. The process for adequate preparation requires various purification conditions are screened and that sufficient quantities of material be scaled up and processed in a time-consuming manner using expensive chromatography equipment. As developments in functional and analytical assays increase throughput and reduce the amount of protein required for analysis, efficient small-volume protein purification would provide high-value information to researchers in earlier stages of drug discovery for both process development and analytical evaluation. Recent advances in the area of miniaturized high-throughput tools for purification, enrichment, and desalting of proteins eliminate bottlenecks associated with traditional protein purification techniques. By performing high-performance functional protein separations on small samples in parallel, it is now possible to obtain more relevant data more quickly, easily, and cost effectively in a completely automated format. Investigation of protein separations in small-scale extraction columns is presented along with optimized conditions enabling functional and analytical characterization of therapeutic proteins purified by this unique format.

Take Home: Small-scale purification columns provide high-performance separation from small sample volumes eliminating the requirement to scale up sample preparation procedures, resulting in reduced sample consumption and time associated with previous purification methods.

Yamuna Dasarathy, director of biopharmaceuticals marketing for Pall Corporation (Port Washington, NY)

Designing Alternative MAb Purification Platforms Without Protein A Affinity By Using Mixed-Mode Chromatography Sorbents

Current platforms for MAb purification — mostly relying on protein A as the first capture step — are robust and efficient but significantly increase downstream purification costs, mainly due to protein A resins. To decrease manufacturing costs, the industry is increasingly looking for purification schemes and new platform processes without protein A affinity resins. Mixed-mode chromatography, an alternative purification platform, offers new selectivity exploiting a combination of both ionic and hydrophobic interactions that can be useful in selectively isolating antibodies from contaminants. In this study, we have characterized interactions between model proteins and three mixed-mode chromatography sorbents (MEP, HEA, and PPA HyperCel from Pall Life Sciences. We selected a panel of proteins with different isoelectric points (pl) and hydrophobicities and developed purification processes at different pH and ionic strength using the above mixed mode sorbents. The optimized MAb purification platform process included very efficient removal of host-cell proteins (HCP) and aggregates as well. Our results show that mixed-mode sorbents when used at capture or intermediate purification step expand the portfolio of options for MAb purification platforms with or without the expensive protein A affinity chromatography step.

Take Home: Current platform processes for MAb purification increase the downstream purification cost because of the expensive protein A step. By optimizing the interactions between proteins and mixed-mode sorbents at different pH and salt concentration, one can selectively purify MAb as well as eliminate host cell protein contaminants and aggregates. Mixed-mode
sorbents used at the capture or intermediate purification step offer an inexpensive alternative to the current MAb platform process

Ludovic Chauchat, biopharma market director in sales for Novasep (Pompey, France)

AbSolute by Novasep: High Performance Protein A Media

The new AbSolute modified silica-based media has been launched for the capture of monoclonal and polyclonal antibodies from different feedstocks. It is based on a modified silica matrix that exhibits strong mechanical, thermal and chemical stability. The rigidity of the matrix combined with a unique kinetic performance allows the use of larger columns and greater bed lengths for operations at higher velocities (≤1,000 cm/h). Optimized particles and pores allow high efficiency and faster mass transfer which increase the dynamic binding capacity (DBC) at high velocities. AbSolute media are stable through repeated cleaning and sanitization steps even with intensive exposure to 50 mM NaOH. This significantly improves productivity.

Take Home: Key benefits of AbSolute media are antibody capture productivity improved by 2–3× compared with industry standards; rigid, monodisperse beads enable the highest linear velocities with a low pressure drop; highest DBC at high linear velocities; easy to pack; stable with repeated alkali washing; linear scale-up from lab to industrial scales; no compromise between bed height and velocity.

Lihua Yang, technical operations scientist at Abbott Laboratories (Worcester, MA)

Development of a Sensitive Assay for MAb and Fragments in Cleaning Validation

Cleaning validation/verification of manufacturing process equipment is required for GMP compliance. The total organic carbon (TOC) assay has traditionally been used to verify the effectiveness of cleaning procedures for removal of product residual. Its detection limit (<0.1 ppm) is sometimes insufficient to monitor the effectiveness of cleaning for low-dose products with a maximum allowable carry over limit below the TOC detection limit. Therefore, a highly sensitive and product-specific assay would be desirable as an alternative or supplementary method to detect residual product per ICH guidelines. This presentation describes the development of ELISA-based assay for the detection of general MAbs and their fragments with a sensitivity 400-fold higher than that of the traditional TOC assay. Assay qualification demonstrates high sensitivity, accuracy, reproducibility, and robustness. Thus, the newly developed MAb assay provides a valuable tool for equipment cleaning validation.

Take Home: To support production cleaning validation, a sensitive assay for detecting monoclonal antibody and the fragments has been developed and implemented.

Jia-li Liao, senior staff scientist in process chromatography R&D at Bio-Rad Laboratories (Hercules, CA)

Nuvia S: A High-Capacity Cation Exchanger for Process Purification of MAbs

The Nuvia S high-capacity strong cation exchange support is designed for optimal bioprocessing performance. It is built on the macroporous rigid UNOsphere base matrix incorporating polymeric surface extenders with optimized density of sulfonate groups. Such a comprehensive approach gives Nuvia S media the excellent dynamic binding characteristics for target biomacromolecules and low backpressure at high flow rates required for downstream processes. We have evaluated the impact of flow rate, load pH, and conductivity on the binding of polyclonal human immunoglobulins G and MAbs with this new chromatographic support. Preliminary studies on the removal of product-related impurities using Nuvia S media were also conducted. Our data indicate that this novel cation exchanger maintains very high binding capacity over a broad range of linear flow rate and variation in load compositions, providing the robustness needed for initial capturing of MAbs from cell culture harvest. The resolution power of Nuvis S support also makes it an outstanding choice for intermediate polishing operation.

Take Home: A new ultrahigh-capacity ion exchanger has been developed with binding capacities for antibodies approaching 200 mg/mL. Data on binding and purification properties of this new support will be presented.

Mark Snyder, manager of the process R&D applications group in process chromatography at Bio-Rad Laboratories (Hercules, CA)

A New Robust Method for Protein Elution from Ceramic Hydroxyapatite

Ceramic hydroxyapatite (CHT) media are used for protein purification owing to its superior removal of all process stream impurities (e.g., aggregates, HCP, and viruses). At commercial scale, they can exhibit fewer cycles than desired. This is due to the release of protons during elution, lowering mobile phase pH and decreasing the stability of CHT. We have developed a novel technology, called the surface neutralization system (SNS), which desorbs protons from the surface of CHT prior to elution. We have demonstrated that the SNS technique extends the useful life of CHT and prevents pH drop during elution, without desorbing the target protein, when that protein is a MAb or polyclonal human IgG. Data will be presented demonstrating that this technology does not affect the common quality outputs usually monitored during antibody purification.

Take Home: A simple, inexpensive method for dramatically extending the robustness of CHT has been developed. Details of how to use this method, and its transparency in terms of CHT purification properties, will be presented.

Andrew Sinclair, president of Biopharm Services (Chesham, UK)

Toward Sustainable Economic Bioprocessing: How Advanced Modelling Provides Insight

The costs associated with the biologics manufacture are a significant and growing proportion of sales prices. In recent years much attention has focused on improving product expression, but it is clear that this only takes us so far in the pursuit of sustainable economic processing. The alternative approach discussed in this poster is to examine in detail the contribution of the downstream processes to the product manufacturing cost. Here we examine the role process models play in quantifying the impact that an increase in upstream product titre has upon the cost of goods (CoG) for a large-scale manufacturing process, particularly with respect to a protein A purification step. Using industry averaged data we highlight how a scalable and flexible process model allows the user to run scenarios in silico to assess the impact of process choices and resin selection upon the feasibility and economic sustainability of the process.

Take Home: Process modeling provides a powerful framework for the rigorous assessment of the impact that innovation can have on product recovery Understanding the costs drivers is important to making informed decisions at the process development scale.

Linda Rich, research scientist in technical operations at Abbott Laboratories (Worcester, MA)

Impact of Acidification of Mammalian Cell Culture Harvest on Process Robustness and Performance

Clarification of high–cell-density mammalian cell culture broths is typically performed with a combination of centrifugat
ion and depth filtration steps, followed by a 0.2-μm filtration step. Acidification of the cell culture broth was evaluated as a means of improving filterability and hence the performance of the clarification process. This presentation describes a robust method toward the acidification of cell culture harvest before cell removal. The study demonstrated that cell culture acidification not only reduces host cell impurities, but also enables significant improvement in filtration throughput of depth and sterile filters, achieving process robustness and a reduction in filter costs. Additionally, acidification markedly improves the product clarity during the holding period between harvest clarification and capture leading to further filter cost reduction.

Take Home: Acidification of harvest reduces host-cell impurities and significantly improves filtration throughput of depth and sterile filters. Therefore, harvest acidification leads to process robustness and reduces filter cost.

Siu-Man (Kelvin) Lau, senior process engineer in technical operations at Abbott Bioresearch Center (Worcester, MA)

Feasibility Study of Continuous Chromatography on MAb Capture Processes

Continuous chromatography using the simulated moving bed concept can improve throughput and productivity for protein purification as well as efficient resin use. To evaluate this technology for monoclonal antibodies (MAb) capture, lab-scale studies were performed using a conventional ÄKTA Explorer 100 system reconfigured with two load lines. Performance of a four-column system for capturing MAbs from clarified cell culture harvest using protein A resin will be presented. The study demonstrated that the conventional ÄKTA Explorer system is capable for evaluating this technology at laboratory scale. Feasibility for MAb capture, in terms of productivity and impurity profile will be presented.

Take Home: Continuous chromatography by simulated moving bed has the promise to alleviate downstream capacity bottleneck, reduce resin and buffer use, and improve productivity.

Jacob Pattasseril, senior engineer in cell therapy R&D for Lonza Walkersville Inc. (Walkersville, MD)

Tangential Flow Filtration: A New Application for Therapeutic Cell Processing

Currently, therapeutic cells are harvested and purified using open centrifugation technology. Open centrifugation is time consuming, increases process risk, and is cost prohibitive to process high-volume batches. We have developed and characterized a scalable, closed system technology based on tangential-flow filtration (TFF) that can process high volume batches in one to three hours while maintaining >90% cell viability, >85% product recovery, and cell functionality. The TFF system includes standard hollow-fiber filters, processing bags and tubing sets, and control pumps. Mesenchymal stem cells (MSCs) and fibroblasts were processed through the TFF system at varying shear rates (1,800–5,500/sec), and samples were obtained at varying processing times and assayed for cell concentration, viability, BSA residual levels, and delayed onset of cell death. Shear rates in the range of 1,800–3,000/sec caused a viability drop ≥3%, whereas 3,100–3,500/sec caused a viability drop of 5–7%. Shear rates >3,500/sec caused viability drops from 12–20% in the processed cells. Viabilities of >90% and product recoveries of >85% were consistently achieved in multiple TFF runs in which shear rates were maintained at <3,000/sec. ViaLight assay results proved that the processed cells had equivalent viability and metabolic activity after 24 hours of culture as unprocessed cells. Scale-up runs at 25 L (10–20 billion cells) were successfully performed with >90% cell viability and >85% product recovery. The diafiltration (cell washing) performed during the TFF process decreased BSA residuals approximately 6,000-fold to less than 100 ng/mL, well below the current FDA requirement of 1 ppm. In conclusion, the fluid shear rate in TFF should be maintained at a narrow range (1,800–3,000/sec) to achieve acceptable product quality parameters such as >90% cell viability and >85% product recovery. This system has been designed as a completely closed system for aseptic processing.

Take Home: TFF is a scalable, single-use technology to process therapeutic cell suspension. Shear rate is a critical process parameter that should be maintained in a narrow range. Uses of TFF instead of existing technologies will decrease processing time by one-to twofold.

Saravanamoorthy Rajendran, associate scientist II in process biochemistry at MedImmune (Gaithersburg, MD)

Identification and Implementation of an Aggregate Removal Step in a Large-Scale Manufacturing Process

This presentation discusses the design of a rapid method to develop an aggregate removal step. This study involved development of a stepwise method to identify and implement an additional purification step to reduce soluble aggregates to <1.0% in a clinical product. The first step involved the isolation of the soluble aggregates using preparative-scale size-exclusion chromatography (SEC) for biochemical and biophysical characterization. The next step involved parallel screening and optimization of chromatography gels representing different types of chromatographic separation modalities using gradient and step elution to determine optimal gel and conditions for aggregate removal. The final step involved process characterization and scale up verification studies before successful implementation in clinical manufacturing. The results from clinical manufacturing demonstrated that the new purification step effectively reduced the soluble aggregates from 3.4% to 0.5%.

Take Home: Rapid process development, optimization, and implementation of an aggregate removal step at large scale in an early stage MAb purification process.

Jeff Wilson, western region sales manager for YMC America.Inc. (Allentown, PA)

Development of Hydrophilic Polymer-Based Ion-Exchange Media for Analysis and Purification of Various Biological Molecules

Increasingly complex separation problems are requiring the development of better analytical and preparative ion-exchange materials for separation of biological molecules. BioPro analytical IEX media has been developed for SAX and SCX separations in reproducible 5-μm porous and nonporous hydrophilic particles, and preparative 30-and 75-μm porous hydrophilic polymer particles — with emphasis on reproducibility, high loading capacity, nonspecific binding, and stability for clean-in-place protocols. Example separations with these media are shown throughout the poster.

Take Home: BioPro QA and SP analytical 5-μm porous and nonporous particles for reproducible high-speed separations. BioPro porous 30-and 75-μm QA and SP particles for reproducible, high loading capacity, nonspecific binding SAX and SCX media with CIP capability for high throughput polishing and capture steps.

Kristina Nilsson-Välimaa, research engineer in R&D at GE Healthcare (Uppsala, Sweden)

Optimization of Intermediate Wash Conditions Using High-Throughput Techniques

A case study investigating different intermediate wash conditions for purification of a MAb on a protein A affinity resin will be presented. The impact of urea, isopropanol, pH, and salt on host-cell protein (HCP) removal was studied using Design of Experiments (DoE). Optimal conditions were identified. High-throughput techniques using 96-well filter plates were applied to design, perform, and evaluate the study. Data obtained using the high-throughput format was verified with a traditional column based format with excellent correlation.

Take Home: Process development with 96-well filter plates reduces sample requirements, speeds up process development, and enables efficient development of intermediate wash conditions.

Ranje
et Patil, process engineer II in the bioscience network of Millipore Corporation (Billerica, MA)

Single-Use Redundant Filtration Assembly: Design and Testing

Due to increased regulatory expectations and valid technical reasons, the use of redundant filtration operations for bulk and final fill operations is becoming increasingly common. Single-use redundant filtration assemblies offer an efficient and flexible alternative to stainless steel redundant filtration systems. Unlike traditional stainless steel systems, a disposable option eliminates the need for CIP and sterilization steps and the associated validation. Preparation time is significantly reduced by single-use assemblies due to the ease of handling and presterilized format. Redundant filtration operations in multiproduct facilities can be performed without spending the extra validation time often required for nondisposable systems. Disposable assemblies are capable of handling the same pressures and flow rates as stainless steel while maintaining similar use sequences. This study identified the best design for redundant filtration operations by utilizing Millipore’s single-use technology products and standardized assembly components. The design was finalized with input from a global Millipore technical and quality team to comply with international regulatory requirements. This study also demonstrated the system capability to withstand high pressure that is used for integrity testing and drying of these assemblies. An optimum use sequence of the preparatory steps was designed and tested. All preparatory steps and filtration operations can be performed on this system without breaching the sterility of the gamma irradiated assembly. Preuse integrity testing was performed on both filters without compromising sterility. With implementation of the Millipak Barrier hydrophilic/hydrophobic filter on the assembly outlet, flush volume limitations due to catch bag and/or can size were eliminated. System specifications such as leachables and extractables, hold-up volume, and flushing requirements were established for a single-use assembly using Opticap XL10 capsules with a 0.22-μm Durapore filter.

Take Home: This study identified the best design for redundant filtration operations with Millipore’s single-use technology products and standardized assembly components. The design was finalized with input from a global Millipore technical and quality team to meet compliance with international regulatory requirements. This study demonstrated the capability of the system to withstand high pressure used during IT testing and drying of these assemblies. An optimum use sequence for preparatory steps was designed and tested. All preparatory steps and filtration operations could be performed on this system without breaching the sterility of the gamma irradiated assembly. Preuse integrity testing was performed on both filters without compromising sterility. With implementation of the Millipak Barrier hydrophilic/hydrophobic filter on the assembly outlet, limitation on flush volume due to catch bag and/or can size was eliminated. System specifications such as leachables and extractables, hold-up volume, and flushing requirements were established for a single-use assembly using Opticap XL10 capsules with a 0.22-μm Durapore filter.

Eric Reese, director of business development for biotherapeutics products in the MicroCal Products Group of GE Healthcare (Northampton, MA)

Differential Scanning Calorimetry: A Valuable Tool for the Development of Biotherapeutic Antibody Manufacturing Processes

It is important for a biotherapeutic to maintain its stability throughout storage until final delivery to a patient. However, many proteins have a propensity to aggregate and precipitate — due to intrinsic factors plus handling and processing steps — which leads to a significant loss in profit. The thermal transition midpoint (T_m) of a biotherapeutic has been shown to be an exceptionally good indicator of the relative stability of biotherapeutics in solution. Differential scanning calorimetry (DSC) allows for the accurate, rapid, and easy measurement of T_m. In addition, DSC can be used to prescreen for the appropriate bioprocessing conditions for maintaining the stability of a biotherapeutic during manufacturing. In this poster, we describe how scientists at Diosynth Biotechnology use thermal stability data obtained by DSC to characterize the stability of an antibody after a low pH viral inactivation hold step followed by pH neutralization. DSC was used to evaluate the excipient effect on protein stability during the transition from low pH to neutral pH. The data from this study are useful for designing and optimizing processes for biopharmaceutical manufacturing.

Take Home: Use thermal stability data obtained by DSC to characterize the stability of an antibody in different solutions/excipients. Data from this study is useful for designing and optimizing processes for biopharmaceutical process development and manufacturing.