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Research ArticleResearch

Retention of Acholeplasma laidlawii by Sterile Filtration Membranes: Effect of Cultivation Medium and Filtration Temperature

Alexander Helling, Hannes König, Felix Seiler, Ralph Berkholz, Volkmar Thom and Milan Polakovic
PDA Journal of Pharmaceutical Science and Technology May 2018, 72 (3) 264-277; DOI: https://doi.org/10.5731/pdajpst.2017.008102
Alexander Helling
1Sartorius Stedim Biotech GmbH, Göttingen, Germany;
2Department of Chemical and Biochemical Engineering, Institute of Chemical and Environmental Engineering, Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Slovakia;
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  • For correspondence: alexander.helling@sartorius-stedim.com
Hannes König
3Department of Medical Engineering and Biotechnology, University of Applied Science Jena, Jena, Germany; and
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Felix Seiler
4Faculty of Natural Sciences, University of Applied Sciences Esslingen, Esslingen, Germany
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Ralph Berkholz
3Department of Medical Engineering and Biotechnology, University of Applied Science Jena, Jena, Germany; and
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Volkmar Thom
1Sartorius Stedim Biotech GmbH, Göttingen, Germany;
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Milan Polakovic
2Department of Chemical and Biochemical Engineering, Institute of Chemical and Environmental Engineering, Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Slovakia;
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Abstract

This experimental study compares cell size, zeta potential, and the ability to penetrate tailor-made size exclusion membrane filters of mycoplasma Acholeplasma laidlawii cultivated in five different cultivation media. The influence of relevant filtration process parameters, in particular transmembrane pressure and filtration temperature, on their respective retention was tested. The impact of the filtration temperature was further evaluated for the Gram-negative bacteria species Brevundimonas diminuta, the Gram-positive bacteria species Staphylococcus epidermidis, the Pseudomonas phage PP7, and the mycoplasma species Mycoplasma orale. The findings were correlated to the different mechanical properties of the particles, especially also with respect to the different bacterial cell envelopes found in those species. This study suggests that mycoplasma, surrounded by a flexible lipid bilayer, are significantly susceptible to changes in temperature, altering the stiffness of the cell envelope. Mycoplasma retention could thus be increased significantly by a decreased filtration temperature. In contrast, Gram-negative and Gram-positive bacteria species, with a cell wall containing a cross-linked peptidoglycan layer, as well as bacteriophages PP7 exhibiting a rigid protein capsid, did not show a temperature-dependent retention within the applied filtration temperatures between 2 and 35 °C. The trends of the retention of A. laidlawii with increasing temperature and transmembrane pressure were independent of cultivation media. Data obtained with mycoplasma M. orale suggest that the trend of mycoplasma retention at different filtration temperatures is also independent of the membrane pore size and thus retention level.

LAY ABSTRACT: Media in biopharmaceutical processes are sterile-filtered to prevent them from bacterial contamination. Mycoplasma represent a relevant class of bacteria. In this publication it is shown that mycoplasma cell size depends on the media they are cultivated in. Membranes used for sterile filtration retain bacteria predominantly by size exclusion. Thus, an altered cell size can result in different retention values. Another characteristic of mycoplasma is the flexible lipid bilayer and the absence of a rigid cell wall. The lipid bilayer can undergo a phase transition from a gel to a liquid-crystal phase at a certain temperature, which makes it stiffer at lower temperatures. A higher stiffness can result in higher retention values during filtration, as the deformability of the mycoplasma cell is lower and the cell does not squeeze through the membrane pores.

Abbreviations: ALCM: A. laidlawii culture medium; ASTM: American Society for Testing and Materials; ATCC: American Type Culture Collection; CFU/mL: colony-forming units per milliliter; DLS: Dynamic light scattering; LRV: Log reduction value; PES: Polyethersulfone; PFU/mL: Plaque-forming units per milliliter; PSD: Particle size distribution; PVP: Polyvinylpyrrolidone; SDS: Sodium dodecyl sulfate; SEM: Scanning electron microscopy; SLB: Saline lactose broth; TMP: Transmembrane pressure; TSB: Tryptic soy broth

  • Bacteria
  • Mycoplasma
  • Microfiltration
  • Retention
  • Phages
  • Transmembrane pressure
  • Filtration temperature
  • Deformation
  • Stiffness
  • Softness
  • © PDA, Inc. 2018
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PDA Journal of Pharmaceutical Science and Technology: 72 (3)
PDA Journal of Pharmaceutical Science and Technology
Vol. 72, Issue 3
May/June 2018
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Retention of Acholeplasma laidlawii by Sterile Filtration Membranes: Effect of Cultivation Medium and Filtration Temperature
Alexander Helling, Hannes König, Felix Seiler, Ralph Berkholz, Volkmar Thom, Milan Polakovic
PDA Journal of Pharmaceutical Science and Technology May 2018, 72 (3) 264-277; DOI: 10.5731/pdajpst.2017.008102

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Retention of Acholeplasma laidlawii by Sterile Filtration Membranes: Effect of Cultivation Medium and Filtration Temperature
Alexander Helling, Hannes König, Felix Seiler, Ralph Berkholz, Volkmar Thom, Milan Polakovic
PDA Journal of Pharmaceutical Science and Technology May 2018, 72 (3) 264-277; DOI: 10.5731/pdajpst.2017.008102
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Keywords

  • Bacteria
  • Mycoplasma
  • Microfiltration
  • Retention
  • Phages
  • Transmembrane pressure
  • Filtration temperature
  • Deformation
  • Stiffness
  • Softness

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