Downstream Processing in Biopharma

Downstream processing (DSP) is the series of unit operations following the completion of cell growth and expansion, and the completed production or synthesis of drug substances or other components. Downstream processing aims to isolate, purify, and concentrate the previously synthesized drug substance (DS) or other product from the bulk growing matrix. Historically, investment has been made to improve yield and titer upstream, making biologics development more economical. In previous years, downstream processes were not addressed at the same level and now require further optimization.

Biopharmaceutical downstream processing refers to the recovery and purification of a drug substance from natural sources, such as animal or bacterial cells. Biopharmaceutical DSP is applicable in monoclonal antibodies (mAb) or protein processes and the manufacture of oligonucleotides, polysaccharides, various vaccines, bioconjugates, gene therapies, and cell therapy products.

Downstream processing may include initial formulation activities, signifying the transition from DS to drug product (DP). Important considerations include managing and measuring product quality attributes, multiple process parameters, sources and quantities of impurities, waste streams, and biohazards.

DSP activities are performed at laboratory-scale process development, pilot, and manufacturing scales and aided by process analytical technology (PAT) and Manufacturing Science and Technology (MSAT) teams focusing on process optimization, scale-up, and troubleshooting. Comparable downstream processing at discovery biology scales or early stages of high-throughput target screening is not as common, although can be performed similarly, in principle.

This guide discusses how scientists utilize process analytical technology (PAT) to transform day-to-day workflows and significantly improve downstream processes. Topics include:

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

• Pre-treatment: 
  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, impurities, and the drug substance. Acid or surfactant lysis is almost exclusively utilized with bacterial or yeast cultures. 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 mass-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, and ionic strength to decrease solubility.
• Centrifugation: Broadly adopted as a method of separation in bioprocessing, centrifugation is a common and major technique used to indiscriminately separate particle debris based on mass or molecular weight as a function of bowl speed, fill, and discharge rates. Common industrial centrifuges remove significant quantities (up to 99%) of particulate mass for particle sizes as small as 5µm, and sometimes as small as 1µm for highly optimized processes. Despite this efficiency, centrifuges can still pass problematic quantities of microparticles, and especially nanoparticles, into downstream operations that will require further processing. With bioprocesses commonly achieving high to very high total biomass (micro and nanoparticles) and variable final viable-cell densities, fixed centrifugation capacities can also limit process throughput and are not widely considered practical for continuous bioprocessing.
  
• Depth Filtration, Hollow Fiber: Depending on the nature of the product and process, harvest methods typically include depth filtration (Fig. 1-1) or hollow fiber filters (Fig. 1-2). With depth filtration, a particle-containing mixture is pumped through a membrane or another interface to separate debris from the 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 before backflushing with counter-flow media. 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 (Fig. 1-3a)
  • Select cell or particle separation from the feed streams into a new capture stream with diversion or separation of a second particle population (Fig. 1-3b)
  • Relocate an entire particle system into a new stream and/or reallocation of fluids, possibly in recirculation (Fig. 1- 3c)
  • Interact hydrodynamically, where a modulated mixture of both particle selection and fluid exchange may occur (Fig. 1-3d)

Inline particle size analyzers, such as ParticleTrack™ with FBRM® technology, measure 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™ with Image2Chords™ 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.

The method and strategy of capture change slightly depending on the nature of the target molecule. 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 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).

Principal measurement goals during chromatography include maximizing the binding capacity of the product to the column, measured as the mass of the product loaded onto the column (column load), minus the mass of the product that escapes the column outlet (known as “breakthrough”). UV is the most common method of product measurement, used extensively for protein and DNA measurement. Alternative methods of product and non-product measurements are also possible. Inline FTIR spectroscopy can be used for quantification and discrimination of components such as surfactants, common sugar or amino acid buffers, lipids, conjugated products, and even products with variable conformation structures, such as those of fragment, monomer, or aggregate forms of mAbs. FTIR is most often used in addition to UV when UV alone is unable to measure important non-protein/non-nucleic acid components.

• 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 selective elution follows a series of wash steps and then releases the target molecule (image c). 
  
  

• 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 (Fig. 2d) while the other components "elute" through the column. IEX is regularly used as an additional or alternative purification method and, therefore, not restricted to capture applications.

The speed of sampling and limit of detection (LoD) with in-situ FTIR spectrometers such as ReactIR™ are advantageous for primary capture chromatography, recording multiple measurement points within quickly eluting peaks or fractions while quantitatively differentiating multiple components. Inline FTIR spectroscopy enables users to 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.

Ultrafiltration (UF) is frequently used in downstream bioprocessing for concentrating a dilute product stream. Ultrafiltration separates molecules within a solution based on the membrane pore size or molecular weight cutoff. Diafiltration (DF) is most often used to exchange the 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 (Fig. 3). 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 path length method.

Several challenges 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 Infrared and Raman spectroscopy enable multiple components to be analyzed simultaneously, with greater precision and dynamic range, and without the delays associated with offline analysis. In-situ FTIR spectroscopy with ReactIR can offer many benefits during buffer exchange:

• Quantify 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

Automated reactors such as EasyMax™ accelerate process development by up to 80% through the capturing of experimental data, accurate control of all critical process parameters (CPPs), and integration of biophysical sensors including pH, conductivity, redox, and dissolved oxygen (DO).

There are several methods employed in sequence that are used to increase the purity of the drug substance (DS). Starting 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 includes polishing chromatography, nano/sterile filtration, crystallization, and viral clearance (Fig. 4).

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 substances, 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 products in a semi-purified matrix. 

Bioconjugate molecules are 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, solvent, or buffer exchange steps throughout (Fig. 5). Biopharmaceutical scientists are adopting technology already used extensively 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. 

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. In-situ FTIR and Raman spectroscopy can provide detailed mechanistic information in real time, eliminating offline delays and sampling inaccuracies.

The goal of formulation is to transition the product molecule from an environment, solvent, or other physical state used to synthesize the product into a form that is acceptable for human clinical administration (Fig. 6). 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 the 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, gene therapies, and 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 the process is obtained, rather than only analyzing the starting and endpoints.

Parallel reactor systems such as EasyMax control all critical process parameters and integrate inline PAT tools such as ReactIR, ReactRaman™, ParticleTrack, EasyViewer, among other biophysical sensors. These technologies are frequently used in formulation to characterize drug substance and drug product stability, final concentration, adjuvant synthesis, polymerizations, encapsulation, adsorptions, and other particulate events.

If you have questions or need help with your technical application, our team of Technical Application Consultants is ready to guide you in the right direction.