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Hydrogenophaga pseudoflava, a diminutive bacterium with an unusual geometry, is used for evaluating passage through 0.2 [micro]m-rated filters and the impact of filtration process parameters and bacterial challenge density. The studies have shown that consistent Hydrogenophaga pseudoflava passage has occurred through 0.2-[micro]m-rated filters, which is in contrast to an absence of passage of nutritionally challenged bacteria that are of similar size but dissimilar geometry.

For drug products manufactured in mammalian cells, safety assurance practices are needed during production to assure that the final medicinal product is safe from the potential risk of viral contamination. Virus filters provide viral retention for a range of viruses through robust, largely size-based retention mechanism. Therefore, a virus filtration step is commonly utilized in a well-designed recombinant therapeutic protein purification process and is a key component in an overall strategy to minimize the risks of adventitious and endogenous viral particles during the manufacturing of biotechnology products. This study summarizes the history of virus filtration, currently available virus filters and prefilters, and virus filtration integrity test methods and study models. There is also discussion of current understanding and gaps with an eye toward future trends and emerging filtration technologies., (© 2021 Wiley Periodicals LLC.)

The presence of aggregates in monoclonal antibody (mAb) drug product (DP) formulations can present product quality challenges. Here we show that use of High Performance Size Exclusion Chromatography (HP-SEC), in conjunction with high-throughput dynamic light scattering (HT-DLS) analyses of mAb DPs can be a useful strategy to determine monomer content and the presence of aggregates under simulated stress conditions. This analytical approach was used to evaluate four commercially available mAb DPs under different conditions i.e.; original formulations, diluted, and thermo-mechanical stressed condition. Due to particle size limitations of HP-SEC columns, resulting in particles accumulating in the column frits prior to reaching the detector for analysis, there is a possibility that large mAb aggregates may not be detected. Both HP-SEC and HT-DLS were able to detect and resolve the mAb monomer (~10-12 nm) of the DPs in their recommended storage conditions. However, the ability to detect large aggregates (>40 nm) by both analytical methods differed, and HT-DLS was able to detect aggregates between 60 nm and 1400 nm under stress conditions. Our data indicates that HP-SEC, in conjunction with HT-DLS, may be beneficial to detect both mAb DP monomer content and multiple aggregate species (1-1000 nm) in the submicron size range., (Published by Elsevier Inc.)

Capture bioprocessing unit operations were previously shown to clear or kill several log 10 of a model mycoplasma Acholeplasma laidlawii in lab-scale spike/removal studies. Here, we confirm this observation with two additional mollicute species relevant to biotechnology products for human use: Mycoplasma orale and Mycoplasma arginini Clearance of M. orale and M. arginini from protein A column purification was similar to that seen with A. laidlawii , though some between cycle carryover was evident, especially for M. orale However, on-resin growth studies for all three species revealed that residual mycoplasma in a column slowly die off over time rather than expanding further. Solvent/detergent exposure completely inactivated M. arginini though detectable levels of M. orale remained. A small-scale model of a commercial low-pH hold step did inactivate live M. orale , but this inactivation required a lower pH set point and occurred with slower kinetics than previously seen with A. laidlawii Additionally, ultraviolet-C irradiation was shown to be effective for A. laidlawii and M. orale inactivation whereas virus-retentive filters for upstream and downstream processes, as expected, cleared A. laidlawii These data argue that M. orale and M. arginini overall would be largely cleared by early bioprocessing steps as shown previously for A. laidlawii, and that barrier technologies can effectively reduce the risk from media components. For some unit operations, M. orale and M. arginini may be hardier, and require more stringent processing or equipment cleaning conditions to assure effective mycoplasma reduction. By exploring how some of the failure modes in commercial antibody manufacturing processes can still eliminate mycoplasma burden, we demonstrate that required best practices assure biotechnology products will be safe for patients., (© PDA, Inc. 2020.)

PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to accelerate immune reconstitution following chemotherapy and is being pursued for biosimilar development. One challenge to overcome in pegfilgrastim biosimilar development is establishing pharmacokinetic (PK) similarity, which is partly due to the degree of PK variability. We herein report that commercially available G-CSF and PEG ELISA detection kits have different capacities to detect pegfilgrastim aggregates that rapidly form in vitro in physiological conditions. These aggregates can be observed using SDS-PAGE, size-exclusion chromatography, dynamic light scattering, and real-time NMR analysis and are associated with decreased bioactivity as reflected by reduced drug-induced cellular proliferation and STAT3 phosphorylation. Furthermore, individual variability in the stability and detectability of pegfilgrastim in human sera is also observed. Pegfilgrastim levels display marked subject variability in sera from healthy donors incubated at 37 °C. The stability patterns of pegfilgrastim closely match the stability patterns of filgrastim, consistent with a key role for pegfilgrastim's G-CSF moiety in driving formation of inactive aggregates. Taken together, our results indicate that individual variability and ELISA specificity for inactive aggregates are key factors to consider when designing and interpreting studies involving the measurement of serum pegfilgrastim concentrations.

Mycoplasma contamination events in biomanufacturing facilities can result in loss of production and costly cleanups. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and may penetrate the 0.2 µm filters often used in the primary clarification of harvested cell culture fluid. Culture cell-based and indicator cell-based assays that are used to detect mycoplasma are highly sensitive but can take up to 28 days to complete and cannot be used for real-time decision making during the biomanufacturing process. To support real-time measurements of mycoplasma contamination, there is a push to explore nucleic acid testing. However, cell-based methods measure growth or colony forming units and nucleic acid testing measures genome copy number; this has led to ambiguity regarding how to compare the sensitivity of the methods. In addition, the high risk of conducting experiments wherein one deliberately spikes mycoplasma into bioreactors has dissuaded commercial groups from performing studies to explore the multiple variables associated with the upstream effects of a mycoplasma contamination in a manufacturing setting. Here we studied the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G1 (IgG1) antibody. We examined M. arginini growth and detection by culture methods, as well as the effects of M. arginini on mammalian cell health, metabolism, and productivity. We compared process parameters and controls normally measured in bioreactors including dissolved oxygen, gas mix, and base addition to maintain pH, to examine parameter changes as potential indicators of contamination. Our work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Importantly, how the M. arginini contamination impacts the CHO cells is influenced by the concentration of CHO cells and rate of perfusion at the time of M. arginini spike. Careful evaluation of dissolved oxygen, pH control parameters, ammonia, and arginine over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before a read-out from a traditional method., (© 2019 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.)

Recent advances in high resolution mass spectrometry (MS) instrumentation and semi-automated software have led to a push toward the use of MS-based methods for quality control (QC) testing of therapeutic proteins in a cGMP environment. The approach that is most commonly being proposed for this purpose is known as the multi-attribute method (MAM). MAM is a promising approach that provides some distinct benefits compared to conventional methods currently used for QC testing of protein therapeutics, such as CEX, HILIC, and CE-SDS. Because MS-based methods have not been regularly used in this context in the past, new scientific and regulatory questions should be addressed prior to the final stages of implementation. We have categorized these questions into four major aspects for MAM implementation in a cGMP environment for both new and existing products: risk assessment, method validation, capabilities and specificities of the New Peak Detection (NPD) feature, and comparisons to conventional methods. This perspective outlines considerations for each of these main points and suggests approaches to help address potential issues.

Continuous bioprocessing holds the potential to improve product consistency, accelerate productivity, and lower cost of production. However, switching a bioprocess from traditional batch to continuous mode requires surmounting business and regulatory challenges. A key regulatory requirement for all biopharmaceuticals is virus safety, which is assured through a combination of testing and virus clearance through purification unit operations. For continuous processing, unit operations such as capture chromatography have aspects that could be impacted by a change to continuous multicolumn operation, for example, do they clear viruses as well as a traditional batch single column. In this study we evaluate how modifying chromatographic parameters including the linear velocity and resin capacity utilization could impact virus clearance in the context of moving from a single column to multicolumn operation. A Design of Experiment (DoE) approach was taken with two model monoclonal antibodies (mAbs) and two bacteriophages used as mammalian virus surrogates. The DoE enabled the identification of best and worst-case scenario for virus clearance overall. Using these best and worst-case conditions, virus clearance was tested in single column and multicolumn modes and found to be similar as measured by Log Reduction Values (LRV). The parameters identified as impactful for viral clearance in single column mode were predictive of multicolumn modes. Thus, these results support the hypothesis that the viral clearance capabilities of a multicolumn continuous Protein A system may be evaluated using an appropriately scaled-down single mode column and equipment., (© 2019 Wiley Periodicals, Inc.)

There is a trend across the pharmaceutical sector toward process intensification and continuous manufacturing to produce small-molecule drugs or biotechnology products. For biotechnology products, advancing the manufacturing technology behind upstream and downstream processes has the potential to reduce product shortages and variability, allow for production flexibility, simplify scale-up procedures, improve product quality, reduce facility footprints, increase productivity, and reduce production costs. On the upstream side of biotechnology manufacturing, continuous perfusion cell cultures are fairly well established. However, truly integrated continuous biomanufacturing requires the uninterrupted connection of continuous unit operations (upstream and downstream) with no isolated intermediate or hold steps occurring between them. This work examines the current scientific and regulatory landscape surrounding the implementation of integrated continuous biomanufacturing., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Metal ions can be enzyme cofactors and can directly influence the kinetics of biochemical reactions that also influence the biological production and quality attributes of therapeutic proteins, such as glycan formation and distribution. However, the concentrations of metals in commercially available chemically defined media can range from 1 to 25,000 ppb. Because such concentration changes can impact cell growth, manufacturing yield and product quality the alteration/fluctuation in media composition should be well controlled to maintain product quality. Here, we describe a platform of analytical methods to determine the composition of several metals in different sample matrices using an advanced automated Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). These methods, validated to ICH Q2R1 regulatory validation parameters, were successfully applied to- (a) screen cell culture media; (b) determine changes in the metal concentration during cell growth in spinner flasks, and, (c) determine effect on the glycosylation pattern and homogeneity of an IgG3:κ produced from a murine-hybridoma cell line in bench-top parallel bioreactors due to a spike in copper and iron concentration. Our results show that maintenance of metal content in the cell culture media is critical for product consistency of the IgG3:κ produced., (Published by Elsevier B.V.)

Stability of therapeutic proteins (TPs) is a critical quality attribute that impacts both safety and efficacy of the drug. Size stability is routinely performed during and after biomanufacturing. Dynamic light scattering (DLS) is a commonly used technique to characterize hydrodynamic size of the TPs. Herein, we have developed a novel method to evaluate in-use and thermal stress stability of TPs using algorithm-driven high-throughput DLS. Five marketed TPs were tested under the guidance of customized algorithms. The TPs were evaluated at relevant temperature conditions as well as under dilution and thermal stress for size stability. We found that the TPs were stable under the in-use conditions tested; however, sample loss due to evaporation can lead to large protein aggregates. A combined assessment of autocorrelation function and photos of sample well could be useful in formulation screening. Dilution of TPs also has an impact on the hydrodynamic size. Thermal stress experiments showed the importance of using different data processing methods to access size distribution. Polydispersity index was useful in evaluating sample heterogeneity. Herein, we show that algorithm-driven high-throughput DLS can provide additional supportive information during and after biomanufacturing and the potential to be used in a quality control environment., (Copyright © 2018. Published by Elsevier Inc.)