Overcoming downstream challenges
These upstream developments create challenges in downstream processing to ensure that the biologics achieve the new quality
and safety specifications. Other challenges for downstream processing include treating high upstream quantities in time, integrating
new chemistries, purifying molecules in a contained system that enables the handling of antibody drug conjugates, and, of
course, ensuring that processes are cost effective. Recent developments in the downstream area, however, are helping to overcome
Process analytical technologies (PAT).
The development of more effective analytical methods has enabled better control over critical quality and quantity attributes
online and atline during downstream processing. FDA encourages the use of these technologies and defines them as "a system
for designing, analyzing, and controlling manufacturing through timely measurements (i.e. during processing) of critical quality
and performances attributes of raw and in-process materials and processes with the goal of ensuring final product quality"
Cycle time, sample preparation, and the nature of the analytical method are key to determining whether on-line or at-line
measurement can be developed. Several other factors also need to be considered when implementing a PAT solution. These factors
include the following: the design of the fluid pathway; communication between the measuring software and process software;
knowledge about the analytical methods' stability, reproducibility, repeatability and precision; linearity; and limits of
quantification combined with processcontrol needs. It also is important to determine whether the techniques should be applied
only in process development or in the production of commercial material as well. Systems for in-line dilution (ILD) of buffers
or clean-in-place (CIP) solutions are examples that have been installed at the industrial scale. The principle of an ILD system
is the preparation of buffers and cleaning solutions by using concentrated stock solutions that are diluted with water to
the desired final concentration. These systems must be robust to compensate for the pressure changes of the water preparation
loops, backpressure from process units (e.g., tangential flow filtration (TFF) or chromatography), and changes in buffer composition
and dilution rates. The principle of ILD is shown in Figure 1.
Figure 1: Principle of in-line dulition. TFF is tangential flow filtration. (ALL FIGURES ARE COURTESY OF THE AUTHOR)
Depending on user requirements, different technical solutions can be used for the dilution itself (e.g.,pumps coupled with
mass flow meters, analogue or proportioning valves) to ensure that the inlet pressure is adequately controlled, and the control
of the composition after the dilution (e.g., conductivity feedback control on the pumps or valves). Cost analysis has shown
that these rather simple solutions can save up to 20% in capital expenditure for new downstream plants (2).
Another example of PAT application concerns atline monitoring and product quality control during the chromatographic elution
of a mixture of lipidic compounds, which ensures that the correct chromatographic fractions are selected. In one particular
case, because the compounds are not ultraviolet active, a light-scattering method was developed to follow the elution profile.
In a second step, an analytical method based on mass spectrometry was implemented to control the composition of the different
lipids. The work commenced with off-line analyses at bench scale and continued with experimental at-line analyses, including
split streams. After the initial laboratory-scale phase, the process and analytical tools were scaled up to find the optimal
technical solution for implementation in an industrial cGMP environment. Figure 2 presents the evaporating lightscattering
detection (ELSD) at-line measurement, coupled with a preparative high-performance chromatography (HPLC) system, which was
used in the study. A very small part of the elution stream that comes from the HPLC is directed to a splitter and transferred
to the light-scattering detector. Because organic solvents are used, a nitrogen purge box is necessary. Using this solution,
the time for in-process sample analysis was reduced to one day, compared with approximately two weeks with initial analytical
methods. The investment and the time of implementation and validatioin need to be balanced with the gain of operational time.
Figure 2: Principle of at-line measurement using evaporating light-scattering detection (ELSD). HPLC is high-performance liquid
The introduction of single-use components into biological production processes started in the early 1970s with liquid filters
and tubings, but by the end of the 1980s, bags also became available for storing small volumes. The beginning of the 2000s
saw the development of fully disposable upstream harvest lines and further development of bufferpreparation systems. Today,
single-use cell culture reactors up to 2000 L and bioreactors up to 500 L are available on the market. Research also is ongoing
to find high-quality, economical solutions for TFF and chromatography single-use systems.
There are many advantages associated with single-use systems, including reduced changeover times (i.e., cleaning, setup, and
storage), reduced cross-contamination risks, availability of fully contained systems, reduction of initial capital expenditure
(in most instances), reduced set-up time, and reduced cleaning validation. These benefits, however, also must be balanced
with other factors, including risk of leachables, compatibility problems, integrity failures, supply-chain interruptions,
scalability limitations, waste management, and operational cost evaluations. A detailed case-by-case evaluation should always
be conducted, particularly for routine production. Figure 3 shows an image of single-use TFF cassettes used for concentration,
diafiltration and the formulation of biologics.
Figure 3: Tangential flow filltration cassettes.
Specific ligands and chromatographic media.
Being able to obtain a high-quality product after only two or three purification steps is a real achievement in the process
development of biologics. However, the process also needs to be scaleable with high productivity. Specific ligands coupled
to rigid chromatographic media for the targeted product can be beneficial in this process. One prominent example is a typical
antibody capture step with Protein A ligands, which can reach up to 90% purity. Ligands can also be developed to polish impurities,
such as residual host cell proteins and transmissible spongiform encephalopathies particles. In the case of polishing, the
capacity is less important, so ligands could be coupled onto membranes to create membrane adsorbers.
With regard to the specificity and stability of the ligand, the structure (i.e., particle size, size distribution, pore size,
pore distribution, mechanical and chemical stability) of the chromatographic beads and the coupling chemistry are also important.
Optimized chromatographic media can increase process performance. In terms of mechanical and chemical stability, pore size,
pore size homogeneity and the coupling chemistry are important for membrane adsorber development.
Productive process dimensioning.
Traditional production processes for biologics are batch based, which means that almost every process flow is collected after
each step in one or several tanks, and often checked for product quality before processing in the next step. Based on development
data, processes could often be more intensive.
In addition to the biopharmaceutical industry, the process intensification phase is mandatory for other industrial biotechnology
sectors such as the processing of antibiotics, amino acids, organic acids and food ingredients. One recent example where a
process migrated from an industrial biotechnology platform to biopharmaceutical production is sequential multicolumn chromatography
(SMCC). This process allows the use of processing time as another dimension; therefore, velocity, resin volume, and the number
of columns can be calculated to fit in a predefined processing time. A significant increase in productivity can be reached
(Examples on Protein A antibody capture showed up to a threefold increase). Buffer and stationary phase consumption was also
cut by at least half (3). This process also can enable continuous downstream processing, where the process steps are connected
to each other to obtain a continuous process flow up to the bulk stage.
Figure 4: Principles of a sequential multicolumn chromatographic (SMCC) process and comparison with a batch one.
Cost studies on monoclonal antibody downstream processing performed by BioPharm Services showed up to a 79% reduction in operational
cost when processes were transformed from batch to continuous ones (4). Additionally, process simulation tools are important
to optimize process dimensioning, production and investment costs, which also can be a support for process characterization
and validation. Figure 4 shows the principles of the SMCC process and compares it with a batch one. Figure 5 shows an industrial
system that enables the operation of SMCC processes.
Figure 5: Example of an industrial system that allows sequential multicolumn chromatography.