Downstream Processing in Biopharma

Biopharmaceutical downstream processing (also known as DSP) refers to the recovery and purification of a drug substance (DS) from natural sources, such as animal or bacterial cells. Biopharmaceutical downstream processing is applicable in mAb or protein processes, as well as in the manufacture of oligonucleotides, polysaccharides and various vaccines.

Downstream processing is typically defined as the unit operations following completion of cell growth and expansion, as well as completion of drug substance synthesis or other product formation. The purpose of downstream processing is to isolate, purify and concentrate the previously synthesized drug substance or other product from the complex bulk matrix.

Downstream processing may also include formulation activities, signifying the transition from drug substance to drug product (DP). Furthermore, downstream processing considerations include resource management and management of multiple waste streams and biohazards. Downstream processing activities are performed at laboratory, pilot and manufacturing scale and involve Process Analytical Technology (PAT) and Manufacturing Science and Technology (MSAT) teams which focus on process optimization, scale-up and troubleshooting. 

Depending on the nature of the product and method of synthesis, Downstream Processing (DSP) generally includes a combination of the following steps:

• Harvest and Filtration
  Harvest is the first step to separate product from bulk debris while optimizing retention of the product yield and quality
  
  
• Primary Capture
  Capture refers to holding onto the desired product while minimizing retention of impurities and by-products. It differs from 'Harvest' in that essentially all bulk debris will have already been removed.
  
  
• Buffer Exchange and Up-concentration
  Ultrafiltration (UF) concentrates a dilute product stream and separates molecules in solution based on the membrane pore size or molecular weight cutoff. Diafiltration (DF) exchanges products from an existing buffer into a new buffer for a following process or a final formulation buffer. Ultrafiltration (UF) and diafiltration (DF) together are the steps of buffer exchange.
  
  
• Purification (and Contaminant or Impurity Clearance)
  Purification removes residual impurities with each successive method employed, while also retaining as much product as possible. The goal is to achieve product purity while minimizing potential yield loss.
  
  
• Bioconjugation (Molecule Dependent)
  Bioconjugate molecules are a new class or generation of biologic molecules which are designed to have an increased efficacy enabled by the combined function of two or more different therapeutic types of molecules. Antibody-Drug Conjugates (ADC) are one of the more common bioconjugates and are synthesized by biochemically modifying an antibody and covalently linking it to another active pharmaceutical ingredient (API).
  
  
• Formulation
  Formulation is the process that transitions a drug substance (DS) into a formulated drug product (DP). Formulation brings the product molecule from an environment, solvent or other physical state into a form suitable for clinical administration.

• Depth Filtration, Hollow Fiber: Depending on the nature of the product and process, harvest methods typically include depth filtration (image 1) or hollow fiber filters (image 2). With depth filtration, a mixture is pumped through a membrane or another interface to separate debris from product in liquid suspension. Hollow fiber filters are used in continuous bioprocessing operations to remove the supernatant expressed drug substance (DS) while temporarily trapping a portion of the biomass in the hollow fiber filter. Acoustic field-flow separation is another emerging technique currently found mostly in development environments. With various modes of operation and configuration, outcomes can:
  • Modulate no cell or particle separation (image 3a)
  • Select cell or particle separation from the feed streams into a new capture stream with diversion or separation of a second particle population (image 3b)
  • Relocate an entire particle system into a new stream and/or reallocation of fluids, possibly in recirculation (image 3c)
  • Interact hydrodynamically, where a modulated mixture of both particle selection and fluid exchange may occur (image 3d)

• Centrifugation: Another broadly adopted method of separation in bioprocessing, however, with the increasingly common trend for high to very high total biomass and moderate final viable cell density, centrifugation is more frequently viewed as a bottleneck and not widely considered practical for continuous bioprocessing.
  
  
• Acid/Surfactant Lysis, Organic-phase Absorption: Product separation with lysis usually pursues a basic goal, effectively disrupt the sequestering structures containing the drug substance or product and create physical separation between any debris or other impurities and the drug substance. Acid or surfactant lysis is almost exclusively utilized with bacterial and occasionally with yeast expression. Similarly, organic-phase absorption and capture of a product is most common among bacterial expression and production systems and is occasionally utilized for yeast systems.
  
  
• Flocculation and Precipitation: These are methods of active particle system manipulation to achieve the goal of separation. Flocculation is generally applied as a method of preparation before filtration or centrifugation. Applying flocculation ensures a high flux over filtration units as well as efficient and cost effective separation of cell material from supernatant. Additional flocculant removal methods and verification protocols may be required. Precipitation is an active particle manipulation triggered by changing parameters, such as solvent, ionic strength to decrease solubility.
  

Particle size distribution (PSD) is known to be a Critical Process Parameter (CPP) involved in filter capacity and is much more impactful than turbidity and pressure alone. Particle Size Distribution (PSD) can be integrated over time to map particle accumulation onto filters (probe above filter) and predict failure. Particle Size Distribution can also be measured below filter to observe trace particulate breakthrough and identify similar events.

The method and strategy of capture changes slightly depending on the nature of the target molecule. Principally, antibodies are captured by affinity resins such as Protein A and Protein G as well as some other selectively engineered methods. Non-antibody proteins and oligonucleotides are more often captured by ion exchange chromatography (IEX). Products such as polysaccharides and complex glycan structures are often captured by Hydrophobic Interaction Chromatography (HIC) or Reversed Phase Chromatography (RPC).

• Affinity Chromatography (Protein A/G) is a first pass chromatographic method (typically called Protein A or ProA) that separates and concentrates the product of interest (typically a mAb) from other soluble materials by reversibly binding or retaining the target protein to the column via specific binding interactions (image a) while the other components "elute" through the column (image b). A series of wash steps followed by selective elution then release the target molecule (image c). There are concerns that arise when using affinity chromatography including, for example, maximizing binding of target protein (column load) to affinity column. Many of these concerns are currently addressed by integrated UV systems which meet current requirements but lack capabilities provided by methods such as in-situ FTIR.
  
  
• Ion Exchange Chromatography (IEX) separates and concentrates the product of interest (proteins, oligonucleotides and others) from other soluble materials by reversibly binding or retaining the target molecule to the column based on the strength of the electrostatic interaction with the solid phase material (image d) while the other components "elute" through the column. Ion Exchange Chromatography (IEX) chromatography is regularly used as an additional or alternative method of purification, therefore not restricted to capture applications.

Ultrafiltration (UF) is frequently used in downstream bioprocessing for concentrating a dilute product stream. Ultrafiltration (UF) separates molecules within a solution based on the membrane pore size or molecular weight cutoff. Diafiltration (DF) is most often used to exchange product into a desired buffer (e.g. from an elution buffer into a final formulation buffer).

• Ultrafiltration and diafiltration (together known as buffer exchange) typically use Tangential Flow Filtration (TFF), where feed flows parallel to the membrane surface rather than perpendicular to the surface. Buffer exchange remains a highly manual operation that is rarely optimized. Product concentration is generally analyzed by a form of UV spectroscopy, either standalone, as a detector for HPLC, or as a variable pathlength method.

There are a number of challenges that arise in the process of buffer exchange:

• Accurate tracking of exchange volumes
• Confident process prediction from non-representative grab samples
• Delayed or bottlenecked response times of offline measurements
• Cumulative product loss with extractive sampling
• Propagation of errors in sample preparation for offline UV and HPLC measurements
• In-situ IR and Raman spectroscopy enable multiple components to be analyzed simultaneously, with greater precision and dynamic range and without the delays associated with offline analysis. 

There are several methods employed in sequence that are used to increase purity of the Drug Substance (DS) beginning with early clarification and extraction steps, followed by an appropriate method of capture and first bulk isolation, subsequently followed by intermediate and then final stages of purification, the latter of which include polishing chromatography, nano/sterile filtration, crystallization and viral clearance.

By eliminating delays associated with offline analysis, in-situ FTIR measurements improve polishing chromatography steps by providing immediate feedback to quantify and speciate fraction components including buffers, drug substance and impurities during intermediate purification steps. This results in improved fraction cuts, aggregate or fragment discrimination and total concentration in real time. For viral clearance, any virus present within the pooled and semi-purified therapeutic suspension is intentionally damaged or abruptly malformed into a non-pathogenic form, usually by altering the environment around the virus. Controlling and refining these Critical Process Parameters (CPP) is enabled by automated reactor platforms with integrated biophysical sensors. Inline Process Analytical Technology (PAT) is useful for characterizing impurities in later stages of purification since concentrations are generally higher and it is easier to distinguish product in a semi-purified matrix. 

Bioconjugate molecules are a new class or generation of biologic molecules, designed to have an increased efficacy enabled by the combined function of two or more different therapeutic types of molecules. Bioconjugate chemistry requires detailed process characterization and optimization. Conventional tools such as microcentrifuge tubes, beakers, hot plates, magnetic stir bars and transfer pipettes are no longer able to meet reproducibility demands around pH, temperature, dosing, mixing and other parameters.

Bioconjugation chemistry relies on a series of well-controlled steps in sequence which may include functional group reduction, activation, API-linker conjugation to the primary biologic and any number of wash or solvent or buffer exchange steps throughout (see diagram below). Biopharmaceutical scientists are adopting methodology used in small molecule R&D, such as EasyMax automated synthesis reactors. EasyMax provides a cohesive architecture so that relevant process parameters including, pH, conductivity, redox, temperature, stirring, dosing, etc. are precisely controlled and experimental data is captured accurately. Automation of bioconjugation chemistry provides data integration, process event correlation, experimental integrity and Design of Experiment (DoE) scale-up parameters. 

The goal of formulation is to bring the product molecule from an environment, solvent or other physical state, into a form that is acceptable for human clinical administration. The product molecule is formulated according to how the final product will be used via inhalation, injection or oral dosing. Long-term stability of the product and associated excipients are assessed to ensure the measured dose and Critical Quality Attributes (CQAs) are within specification following processing, storage and shipping. Besides sterility, ensuring removal of impurities and endotoxins and preventing Drug Product (DP) degradation is essential in maintaining safety and efficacy during the manufacture and long-term storage of a protein therapeutic.

Formulated drug products include proteins (specifically mAb), polysaccharides, nanoparticle systems, organics, oligonucleotides and the many types of vaccines. Certain vaccines will be formulated with an adjuvant, typically an aluminum-based particulate, or an organic emulsion. Vaccine formulation and adjuvant synthesis in particular are workflows well positioned to advantage automated parallel reactor workstations, workflow digitalization and orthogonal Process Analytical Technology (PAT) integration. With real-time PAT, far more knowledge about what is occurring during the process is obtained, rather than only analyzing the starting and endpoints.

• Identify factors contributing to bottlenecks and impurity formation during bioprocess development
• Understand how process conditions impact product concentration and yield
• Minimize inaccuracies and time delays arising from traditional, less efficient production and analysis techniques
• Monitor specific operations such as purification, chromatography, buffer exchange, up-concentration and formulation
• Use data mining methods to fully characterize the relationship between the various downstream processing steps and using this actionable information to optimize performance of continuous processes
• Implement an integrated Quality by Design (QbD)/Process Analytical Technology (PAT) strategy

This guide discusses how scientists are utilizing Process Analytical Technology (PAT) to transform day-to-day workflows and make significant downstream process improvements. Topics include:

• Protein concentration monitoring
• Buffer exchange process optimization
• Vaccine adjuvants composition, distribution and morphology
• Automation of viral inactivation
• Bioconjugation process development

The biopharmaceutical industry is adopting new technology to achieve greater process insight by successfully applying continuous processing methods and enabling comprehensive data capture and electronic record keeping. Via automated laboratory reactors, in-situ spectroscopic analyzers, particle characterization tools and advanced software, Process Analytical Technology (PAT) allows precision control and comprehensive understanding for optimization of downstream operations.

• Harvest and Filtration: ParticleTrack (FBRM) measures particle accumulation and size distribution of cell broth feed going into or escaping filters and can provide capacity correlation or feedback control to minimize process disruptions. EasyViewer performs image analysis and extracts particle accumulation, size distribution and morphology from time-resolved inline images of the process. Placed above or below filters, EasyViewer can provide capacity correlation or feedback control, but is most valuable when applied for root cause analysis, characterization and morphology.
  
  
• Primary Capture: The speed of sampling and LoD for ReactIR are advantageous for primary capture chromatography, recording multiple measurement points within fast events while quantitatively differentiating multiple components. Monitor variable feed inputs, as well as resin quality and lifetime performance. Identify and fingerprint eluate components and eliminate delays in receiving analytical results to make data-driven decisions in real time.
  
  
• Buffer Exchange and Up-concentration: ReactIR captures and quantifies multiple excipients and Drug Substance (DS) species simultaneously in real time. Control buffer usage; monitor membrane effects; use direct in-situ concentration rather than diavolume approximations; increase concentration accuracies from +/-5 % to +/-2 %; eliminate analytical delays. EasyMax automated reactors accelerate process development by up to 80 % through capture and structure of experimental data, accurate control of all Critical Process Parameters (CPP) and integration of biophysical sensors such as pH, conductivity, redox and Dissolved Oxygen (DO)
  
  
• Purification: ReactIR is advantageous for supporting polishing chromatography. Quantify and speciate fraction components including buffers, drug substance (DS) and impurities. Improve fraction cuts, aggregate or fragment discrimination and total concentration in real-time; cut out delays in receiving offline analytical results. With EasyMax applied to viral inactivation, quickly assess DoE space including mixing conditions, temperature control, dosing strategies and rates. Mitigate quality affects of pH-induced deamidation and denaturation. Accelerate process development via integration of biophysical sensors such as pH, conductivity, redox, and dissolved oxygen. With ParticleTrack (FBRM) and EasyViewer the impact of process parameters on crystallization is determined for fast process optimization.
  
  
• Bioconjugation (Molecule Dependent): EasyMax enables quick assessment and fine CPP control of DoE space including mixing conditions, temperature control, dosing strategies and rates. Eliminate experimental variability associated with offline, manual methods and poor critical process parameter controls. ReactIR and ReactRaman provide detailed mechanistic information in real time, eliminating offline delays and sampling inaccuracies.
  
  
• Formulation: EasyMax controls all critical process parameters and integrates ReactIR, ReactRaman, ParticleTrack, EasyViewer and numerous biophysical sensors. Characterize drug substance and drug product stability, final concentration, adjuvant synthesis, polymerizations, encapsulation, adsorptions and other particulate events.

EasyMax automated reactor is a complete workstation for fundamental parameter optimization in downstream processing (DSP) method design and execution. This fully automated system enables precise control over key bioprocessing variables.

• Manage all Critical Process Parameters (CPPs) including reagent and source material identification, dosing, temperature, mixing, pH measurement and titration
  
  
• Readily integrates with in-situ analysis methodology including ReactIR (FTIR), ReactRaman (Raman), ParticleTrack (FBRM), EasyViewer (in-situ image analysis) and EasySampler. Run experiments when unattended; execute intelligent automated sampling when offline analysis is required.
  
  
• iC Software enables automated execution and control of all experiments and peripheral equipment as well as full data capture and record keeping

ReactIR uses mid-infrared spectroscopy to measure both aqueous and organic-soluble components in a vessel or flowing process stream both in-situ and in real time. ReactIR measurements are not impacted by suspended solids, bubbles or cell mass.

• Monitor multiple product quality attributes in-situ. Eliminate delays associated with grab samples
  
  
• Quantify the impact of processing steps on product yield and process efficiency
  
  
• Provides actionable data analytics for real-time decision making and control
  
  
• Supports ICH regulatory guidelines through data-rich experimentation and digitalization
  
  
• Aids in scale-up from R&D to manufacturing
  
  
• Measures analyte concentrations as low as 0.5 mg/mL in volumes from few milliliters to thousands of liters

ParticleTrack and EasyViewer enable scientists to:

• Continuously monitor particles and particle structures, as experimental conditions vary
  
  
• Track batch growth of biological systems in real time with no sample removal required
  
  
• Characterize particle systems conditions going into or escaping operations such as filtration
  
  
• Observe particle mechanisms such as adjuvant synthesis, antigen-adjuvant adsorption and stability 

ParticleTrack with Focused Beam Reflectance Measurement (FBRM) technology is a probe-based particle analyzer that is inserted directly into a vessel to track changing particle size and count in real time. Uses Chord Length Distribution measurements to obtain Particle Size Distribution (PSD). 

EasyViewer is an optical, probe-based instrument that is inserted directly into a vessel to capture images of the particle system. EasyViewer performs image analysis and extracts particle accumulation, size distribution and morphology from time-resolved inline images of the process allowing populations to be trended over time. 

Gregory Dutra, Daniel Komuczki, Alois Jungbauer, Peter Satzer, “Continuous capture of recombinant antibodies by ZnCl₂ precipitation without polyethylene glycol”, Engineering in Life Sciences (2020), 20, 265–274.

The authors report using divalent cations, specifically ZnCl₂, to capture and purify monoclonal antibodies in a precipitation-based method.  Because of the crosslinking nature of divalent cations and eliminating PEG, viscosity from the supernatant and resolubilization dilution factors were very low.

By holding the ZnCl₂ concentration static and varying the pH using an EasyMax automated lab reactor, the authors quantified the effect of pH on yields. The precipitation was monitored with a ParticleTrack FBRM probe and the size distribution of protein clusters was measured at the different pH.  They found that the optimum pH for the precipitation is around pH 6 and 7, and that pH has a great impact on the size of the precipitate clusters, with the largest clusters associated with better yields. They state that FBRM inline monitoring can be used to estimate the resulting yields without offline measurement of the actual concentration during precipitation.

Carmen Mei, Sasmit Deshmukh, James Cronin, Shuxin Cong, Daniel Chapman, Nicole Lazaris, Liliana Sampaleanu, Ulrich Schacht, Katherine Drolet-Vives, Moriam Ore, Sylvie Morin, Bruce Carpick, Matthew Balmer, Marina Kirkitadze, ”Aluminum Phosphate Vaccine Adjuvant: Analysis of Composition and Size Using offline and In-Line Tools”, Computational and Structural Biotechnology Journal (2019), 17, 1184-1194.

Since the interaction between the antigen and adjuvant is important to vaccine immunogenicity, in this research the authors investigated the physicochemical properties of the adjuvant, in this case AlPO₄, such as particle size and chemical composition. They used both offline techniques such as Fourier-Transform Infrared (FTIR) and Raman spectroscopy, X-ray Photoelectron Spectroscopy (XPS), laser diffraction and in-line methods including in-situ FTIR-ATR (ReactIR), Raman (ReactRaman), and Focused Beam Reflectance Measurement (FBRM, ParticleTrack). With respect to the inline measurements, particle size distribution of aluminum phosphate and adsorbed protein were examined by FBRM and compositional analysis of aluminum phosphate was performed using the in-situ ATR probe.

The authors concluded that inline PAT effectively monitors particle size and chemical composition for the various stages of adjuvant manufacturing.  Similar approaches can be utilized to help assess lot-to-lot consistency, as well. They state that the use of in-line PAT supports advanced manufacturing strategies such as real-time product release testing.

Below is a selection of recent publications with downstream processing in biotechnology and the biopharmaceutical industry.

• Gregory Dutra, Daniel Komuczki, Alois Jungbauer, Peter Satzer, “Continuous capture of recombinant antibodies by ZnCl2 precipitation without polyethylene glycol”, Engineering in Life Sciences (2020), 20, 265–274.
  
• Dhanuka P. Wasalathanthri, Matthew S. Rehmann, Yuanli Song, Yan Gu, Luo Mi, Chun Shao, Letha Chemmalil, Jongchan Lee, Sanchayita Ghose, Michael C. Borys, Julia Ding, Zheng Jian Li, "Technology outlook for real‐time quality attribute and process parameter monitoring in biopharmaceutical development—A review", Biotechnology and Bioengineering (2020), 177(10), 3182-3198.
• Carmen Mei, Sasmit Deshmukh, James Cronin, Shuxin Cong, Daniel Chapman, Nicole Lazaris, Liliana Sampaleanu, Ulrich Schacht, Katherine Drolet-Vives, Moriam Ore, Sylvie Morin, Bruce Carpick, Matthew Balmer, Marina Kirkitadze, "Aluminum Phosphate Vaccine Adjuvant: Analysis of Composition and Size Using Off-line and In-Line Tools”, Computational and Structural Biotechnology Journal (2019), 17, 1184-1194.
• Julian Kager, Vladimir Berezhinskiy , Robert Zimmerleiter, Markus Brandstetter, Christoph Herwig, “Extension of a Particle Filter for Bioprocess State Estimation using Invasive and Non-Invasive IR Measurements”, Computer Aided Chemical Engineering (2019), 46, 1417-1422.
• Matthias Brunner, Philipp Brosig, Monika Losing, Marco Kunzelmann, Amandine Calvet, Fabian Stiefel, Jan Bechmann, Andreas Unsoeld, Jochen Schaub, “Towards robust cell culture processes—Unraveling the impact of media preparation by spectroscopic online monitoring”, Engineering in Life Sciences (2019), 19, 666-680.
• Marine Deshors, Olivier Guais, Virginie Neugnot-Roux, Xavier Cameleyre, Luc Fillaudeau, Jean Marie Francois, "Combined in situ Physical and ex-situ Biochemical Approaches to Investigate in vitro Deconstruction of Destarched Wheat Bran by Enzymes Cocktail Used in Animal Nutrition”, Frontiers in Bioengineering and Biotechnology (2019), 7(158).
• Steffen Großhans, Matthias Rüdt, Adrian Sanden, Nina Brestrich, Josefine Morgenstern, Stefan Heissler, Jürgen Hubbuch, “In-line Fourier-transform infrared spectroscopy as a versatile process analytical technology for preparative protein chromatography", Journal of Chromatography A, (2018), 1547, 37-44.
• Anja Lemoinea, Frank Delvigneb, Anika Bockischa, Peter Neubauera, Stefan Junne, “Tools for the determination of population heterogeneity caused by inhomogeneous cultivation conditions”, Journal of Biotechnology (2017), 251, 84-93.
• Anouk Dirksen, Keith A. Davis, Joe T. Collins, Keshab Bhattacharya, Jari I. Finneman, Erin L. Pepin, Jeffrey S. Ryczek, William B. Wellborn, Ratish Mangalathillam, Brad P. Evans, Mark J. Pozzo, Rory F. Finn, “Process development of a FGF21 protein–antibody conjugate”, Peptide Science (2017), 110:e23042.
• Cosima Koch, Andreas E. Posch, Christoph Herwig, Bernhard Lendl, “Comparison of Fiber Optic and Conduit Attenuated Total Reflection (ATR) Fourier Transform Infrared (FT-IR) Setup for In-Line Fermentation Monitoring”, Applied Spectroscopy (2016), 70(12), 1965-1973.
• Andrew Salazar, Julia Bleifuß, Anke Simon, Sabrina Schüßler, Michael Keusgen, Nikolai Stankiewicz, Jörg von Hagen, “Compaction of chemically defined cell culture media increases its dissolution rate through an increase of solvent accessible surface area”, Powder Technology (2016), 301, 110-117.
• Patricio Lopez-Exposito, Angeles Blanco Suarez, Carlos Negro, “Estimation of Chlamydomonas reinhardtii biomass concentration from chord length distribution data”, Journal of Applied Phycology (2016), 28, 2315–2322.
• Christian Sieblist, Marco Jenzsch, Michael Pohlscheidt, “Equipment characterization to mitigate risks during transfers of cell culture manufacturing processes”, Cytotechnology (2016), 68(4), 1381–1401.
• Cosima Koch, Markus Brandstetter, Patrick Wechselberger, Bettina Lorantfy, Maria Reyes Plata, Stefan Radel, Christoph Herwig, Bernhard Lendl, “Ultrasound-Enhanced Attenuated Total Reflection Mid-Infrared Spectroscopy In-Line Probe: Acquisition of Cell Spectra in a Bioreactor”, Analytical Chemistry (2015), 87, 2314−2320.
• Janosch Fagaschewski, Daniel Sellin, Charles Wiedenhöfer, Sven Bohne, Hoc K. Trieu, Lutz Hilterhaus, “Spatially resolved in situ determination of reaction progress using microfluidic systems and FT-IR spectroscopy as a tool for biocatalytic process development”, Bioprocess Biosystems Engineering (2015), 38, 1399–1405.
• Huiquan Wu, Erik Read, Maury White, Brittany Chavez, Kurt Brorson, Cyrus Agarabi, Mansoor Khan, “Real time monitoring of bioreactor mAb IgG3 cell culture process dynamics via Fourier transform infrared spectroscopy: Implications for enabling cell culture process analytical technology”. Frontiers of Chemical Science and Engineering (2015), 9, 386–406.
• Cosima Koch, Andreas Posch, Héctor Goicoechea, Christoph Herwig, Bernhard Lendl, “Multi-analyte quantification in bioprocesses by Fourier-transform-infrared spectroscopy by partial least squares regression and multivariate curve resolution”, Analytica Chimica Acta (2014), 807, 103-110.
• Cornelia Walther, Sabrina Mayer, Alois Jungbauer, Astrid Dürauer, “Getting ready for PAT: Scale up and inline monitoring of protein refolding of Npro fusion proteins”, Process Biochemistry (2014), 49(7), 1113-1121.
• Anne Tscheliessnig, Peter Satzer, Nikolaus Hammerschmidt, Henk Schulz, Bernhard Helk, Alois Jungbauer, “Ethanol precipitation for purification of recombinant antibodies”, Journal of Biotechnology (2014), 188, 17-28.
• Patrick Sagmeister , Magdalena Kment , Andrea Meitz , Timo Langemann, Christoph Herwig, “Soft-sensor assisted dynamic investigation of mixed feed bioprocesses”, Process Biochemistry (2013), 48(12), 1839-1847.
• Patrick Sagmeister, Timo Langemann, Patrick Wechselberger, Andrea Meitz, Christoph Herwig, “A dynamic method for the investigation of induced state metabolic capacities as a function of temperature”, Microbial Cell Factories (2013), 12, 94.
• John Dahlbacka , Kristiina Kiviharju, Tero Eerikäinen, Kaj Fagervik, “Monitoring of Streptomyces peucetius cultivations using FTIR/ATR spectroscopy and quantitative models based on library type data”, Biotechnology Letters (2013), 35, 337–343.
• John Dahlbacka, Jan Weegar, Niklas von Weymarn, Kaj Fagervik, “On-line measurement of the substrate concentrations in Pichia pastoris fermentations using FT-IR/ATR”. Biotechnology Letters (2012), 34, 1009–1017.
• Daniel Landgrebe, Claas Haake, Tim Höpfner, Sascha Beutel, Bernd Hitzmann, Thomas Scheper, Martin Rhiel, Kenneth F. Reardon, “On-line infrared spectroscopy for bioprocess monitoring”, Applied Microbiology and Biotechnology (2010), 88, 11–22.
• Michal Dabros, Moira Monika Schuler, Ian W. Marison, “Simple control of specific growth rate in biotechnological fed-batch processes based on enhanced online measurements of biomass”, Bioprocess Biosystems Engineering (2010), 33(9), 1109-1118.
• Jakob J. Müller, Markus Neumann, Paul Scholl, Lutz Hilterhaus, Marrit Eckstein, Oliver Thum, Andreas Liese, “Online Monitoring of Biotransformations in High Viscous Multiphase Systems by Means of FT-IR and Chemometrics”, Analytical Chemistry (2010), 82(14), 6008–6014.