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March 27, 2018

Simplified Small-Scale Harvest of CHO Cells for mAb Analytics

Comparison of Clarification Principles: Centrifugation & Diatomaceous Earth

Simplified Small-Scale Harvest of CHO Cells for mAb Analytics

Figure 1. Scanning electron microscopy (SEM) picture of calcined diatomaceous earth (DE) with a permeability of 150–300 mDarcy.

  • Abstract

    In this study, it was tested whether the body-feed filtration method using diatomaceous earth (DE) as filter aid lead to different monoclonal antibody (mAb) and filtrate characteristics, in regards to five parameters analyzed compared to the well-established centrifugation method. The parameters investigated were reduction of turbidity, recovery of mAbs, mAb molecular weight, charge heterogeneity, glycosylation pattern, and over all work efficiency.

    The heterogeneous sample pool comprised of various mAb products (two IgG1, IgG2, fc fusion protein, and bispecific antibody) and different cultivation conditions with cell densities between 38.3 x 105 and 163.6 x 105 cells/mL, turbidities between 557 and 1431 NTU and protein titers between 0.17 and 8.84 mg/mL.

    All samples were harvested by both the body-feed filtration method using the Sartoclear® Dynamics Lab P15 kit and by the traditional centrifugation method, followed by sterile filtration. As a result, it could be shown that both cell culture harvest methods lead to similar results in all parameters but one. While leading to high recoveries of unaltered mAbs as well as sufficient turbidity reductions of the cell culture fluid (CCF), the DE method reduced the processing time by more than half.

  • Introduction

    mAbs have been used successfully for years as therapeutic agents for different pathologies, e.g., various types of cancer and autoimmune diseases. They have become one of the main growth drivers of the pharmaceutical industry with a market size of $56.4 billion in 2012 and which is expected to reach $122.6 billion by 2019.1

    However, antibody treatments are significantly more cost-intensive compared to drug therapies with chemically defined small molecules. This is due to the elaborate development and production processes associated with mAbs. The reduction of costs per treatment course is becoming increasingly important because of the continuous addition of new therapeutic antibodies while the budgets of national healthcare systems are simultaneously being limited.2

    As a result, attempts are being made both to increase the yield of antibodies per production volume and to reduce the use of capital investments through shortened development periods.3 Cost reduction can often be achieved by continuous optimization of the individual steps.

    The current study focuses on harvesting CHO cell cultures as well as on the quality of the yielded filtrate for analytical purposes. We present the use of DE as filter aid within a body feed clarification. The sample volumes considered are up to 50 mL, a typical scale for devices like spinner tubes, shaking flasks, and micro-bioreactors such as ambr15, a common setting for cell-line development, early process development, and media optimization.5,6

    The method of harvesting plays an important role with regard to the examination of antibody-specific quality attributes because cellular material has to be removed adequately and antibodies as analytes can be influenced by the clarification process, e.g., by adsorption to filter material.

    The conventional method of harvesting consists of a centrifugation step in which particles (cells, debris, etc.) with high density or large size are separated. A subsequent microfiltration step, where suspended particles are removed from the cell culture fluid (hereinafter referred to as “centrifugation”), is carried out.

    The body-feed filtration method presented here enables the separation of complete cells and coarse debris by using DE while suspended particles are removed by microfiltration at the same time.

    In the following, we demonstrate that the centrifugation and the body-feed filtration method are comparable with regard to particle depletion, mAb recovery, charge heterogeneity, and glycosylation pattern. Additionally, this work shows the efficiency of work for both methods. A heterogeneous sample pool with different types of mAb and cultivation methods was compiled to obtain a comprehensive statement.

  • Body-Feed Filtration by Diatomaceous Earth

    Click Image To Enlarge +
    Figure 2. Principles of clarifying cell cultures by using the conventional (centrifugation) and the body-feed filtration (DE) method utilized by Sartoclear Dynamics® Lab.

    Sartoclear Dynamics Lab products base their filtration principles on body-feed filtration where a filter aid is used to form a porous cake with the suspended solids in a dead-end filter. In a conventional filtration, the cake layer solely formed by biomass is compressible and becomes rapidly impermeable, which leads to filter clogging (Figure 2, left). In contrast to that, the filter aid applied for body-feed filtration creates a nearly incompressible filtration cake, which stays porous and prevents blockage over the complete filtration (Figure 2, right).

    Inspired by the blood and plasma industry, all Sartoclear Dynamics Lab Products operate in body-feed mode using DE as filter aid. The use of DE as a filter aid in fractionation of human plasma was reported for the first time in the middle of the last century7.

    Today DE is well-established as filter aid and in 2015, 75 % of the U.S. diatomite production was produced for filtration application8.

    DE is gained from different species of extinct and living diatom algae, where their fossilized skeleton deposited on lakebeds consist of nearly pure amorphous silica (SiO2).9 The wet mixture is dried and clarified via calcination up to 1,000°C or flux-calcination up to 1,200°C to remove organics and partially volatilized metal ions, while the temperature treatment impacts the porosity of the gained particle agglomerates (Figure 1).

    For biopharmaceutical applications a high level of purity of the DE is required. The Celpure® DE (Advanced Minerals Corporation) family is highly purified DE, where impurities are removed before calcination, making this purity grade specially designed for cGMP pharmaceutical processing.

  • Methods and ResultsCHO Cell Culture and Target Proteins

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    Table 1. Overview of cultivation system, mAb product, turbidity of the cell culture fluid (CCF) and cell culture fluid harvested by centrifugation and by DE, and relative reduction of turbidity by centrifugation and by DE.

    To create stable cell lines, CHO DG44 cells were transfected by electroporation and cultivated under selective conditions for three weeks followed by an amplification step with 30 nM MTX for an additional three weeks. Stable individual cell pools were then expanded and clones were generated by FACS. Clones were analyzed for growth performance and product concentration via fed-batch studies and genetic stability was evaluated during nine-week stability studies, including stability fed-batches (initiated at t = 2 weeks, t = 5 weeks, and t = 9 weeks, respectively).

    In 11 cultivation batches, seven combinations of target proteins and cultivation methods were used (Table 1). In addition to 125 mL and 1,000 mL shaking flasks (SF), 5-L stirred bioreactors (UniVessel, Sartorius) were applied. As target proteins, CHO cell lines expressing different types of antibodies or antibody-derived products (IgG1, IgG2, Fc fusion, bispecific antibody) were selected. Cell density and viability were examined with a Vi-CELL XR system from Beckman Coulter.

  • Cell Culture Harvest

    All cell culture batches (CCF) were harvested after 14 days and clarified in parallel case by centrifugation and DE method. For the clarification, sample volumes between 19 and 31 mL were used.

    The cell culture samples (approx. 30 mL, Falcon tubes) were centrifuged for 5 min at 4,500 g and 20°C. Subsequently, the supernatant was filtered with a syringe filter (Minisart Highflow, 16532-GUK, Sartorius) with a pore size of 0.2 μm. Depending on the sample, a varying number of filters (1–3 devices) had to be used due to blockages.

    The cell culture samples were clarified with the Sartoclear Dynamics Lab P15 (Sartorius). For this purpose, the cell culture suspension was taken with the syringe prefilled with DE and, after a short resuspension, filtered. The P15 is a kit comprising a 20 mL syringe prefilled with DE powder and a syringe filter equipped with a high-purity quartz microfiber prefilter and a 0.2 μm final filter made of polyethersulfone. The filling tube is useful for harvesting cell-culture suspension from ambr15 bioreactors.

  • Turbidity and Particle Depletion

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    Table 2. Overview of cultivation system, mAb product, viable cell concentration (VCC) after 14 days cultivation, mAb titer for the cell culture fluid (CCF) and the clarified cell culture fluid (centrifugation and DE), and recovery rate (centrifugation and DE).

    The centrifugation and DE method was investigated for its ability to remove cell components from the CCF. For this purpose, we determined the turbidity values before and after clarification (Table 1) by the TurbiCheck WL turbidimeter (Lovibond, white light source). To remain in the linear measuring range between 1 and 1,100 NTU, the samples were diluted with one-fold DPBS before turbidity measurement. Afterwards, the reduction of turbidity was determined by calculation of the rato of values from harvested and unharvested samples.

    mAb Protein Titer and Recovery

    The mAb titer was determined in the CCF before and after the harvest procedure using the Octet QKe system equipped with the protein A biosensor (ProA) from FortéBio without any interfering sample preparation. Due to the diversity of samples, titers were achieved in a range of 0.17 to 8.84 g/L prior to harvest. Table 2 shows the measured titers and calculated recovery values.

  • Glycosylation Pattern, Charge-Heterogenity, and Protein Size

    The clarified samples were purified by means of Protein A using HP Spin Trap Columns or PreDictor MabSelect SuRe plates from GE Healthcare. For this purification step we used 1 x PBS as binding buffer, 100 mM citric acid, pH 3.0 as elution buffer and 1 M TRIS-HCl pH 9.0 as neutralization buffer.

    Subsequently, the buffer was replaced by WFI using an ultrafiltration device (Vivaspin 500, 10 kDa from Sartorius). The NanoPhotometer® N 50 of Implen was used to adjust the protein concentration for the following investigations.

    To determine the carbohydrate structure, charge heterogeneity and molecular weight of the targeted mAbs the LabChip GXII Touch 24 from Perkin Elmer was used. The following assays were used for the respective attributes.

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