ASTM E3326-22

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3326 − 22
Standard Guide for
Application of Continuous Manufacturing (BioCM) in the
Biopharmaceutical Industry
This standard is issued under the fixed designation E3326; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope free from external biological impurities and microbial contami-
nation (for example, bioburden, viruses, and mycoplasma) is
1.1 This guide is intended as a complement to Guide E2968.
critical.
It provides key concepts and principles to assist in the
appropriate selection, development, and operation of continu- 1.8 This guide is intended to apply in both the development
ous processing technologies for the manufacture of biologi- of a new process or the redesign of an existing one.
cally derived products.
1.9 A manufacturer may choose to implement continuous
1.2 Several of the principles covered in Guide E2968 are manufacturing for discrete unit operations in stages as they
applicable to biomanufacturing. However, processes for bio- develop process understanding before implementing a fully
logically derived products differ from those for synthetic drugs connected or continuous manufacturing process.
in a number of fundamental ways in addition to their source
1.10 Units—The values stated in SI units are to be regarded
(for example, format: aqueous liquids versus powders; scope:
as the standard. No other units of measurement are included in
genesis to final formulation). This guide is intended to provide
this standard.
greater clarity for biomanufacturing. It does not imply that
1.11 This standard does not purport to address all of the
topics in Guide E2968 that are not covered here do not apply
safety concerns, if any, associated with its use. It is the
to continuous manufacturing (CM) for biologics.
responsibility of the user of this standard to establish appro-
1.3 Biologically derived products also differ widely from
priate safety, health, and environmental practices and deter-
each other in terms of modalities, source materials, and the
mine the applicability of regulatory limitations prior to use.
manufacturing technologies used, not all of which are equally
1.12 This international standard was developed in accor-
amenable to operating in a continuous mode.
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
1.4 Opportunities do exist for the introduction of continuous
Development of International Standards, Guides and Recom-
technologies, for example, efforts are ongoing to adapt pro-
mendations issued by the World Trade Organization Technical
cesses for large-scale manufacture of broadly applicable mo-
Barriers to Trade (TBT) Committee.
dalities such as monoclonal antibodies to a continuous format.
This guide is intended to provide guidance to the design and
2. Referenced Documents
implementation of antibody processes.
2.1 ASTM Standards:
1.5 The principles can be applicable to unit operations or
E2363 Terminology Relating to Manufacturing of Pharma-
processes or both for other modalities but may not be appli-
ceutical and Biopharmaceutical Products in the Pharma-
cable to all bioprocesses.
ceutical and Biopharmaceutical Industry
1.6 Particular consideration should be given to the develop-
E2475 Guide for Process Understanding Related to Pharma-
ment and application of the appropriate scientific understand-
ceutical Manufacture and Control
ing and engineering principles that differentiate CM from
E2537 Guide for Application of Continuous Process Verifi-
traditional batch manufacturing.
cation to Pharmaceutical and Biopharmaceutical Manu-
1.7 Since much of the processing is done under conditions
facturing
amenable to microbial growth, maintaining process streams
E2888 Practice for Process for Inactivation of Rodent Ret-
rovirus by pH
This guide is under the jurisdiction of ASTM Committee E55 on Manufacture
of Pharmaceutical and Biopharmaceutical Products and is the direct responsibility of For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Subcommittee E55.12 on Process Applications. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Current edition approved Sept. 1, 2022. Published October 2022. DOI: 10.1520/ Standards volume information, refer to the standard’s Document Summary page on
E3326-22. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3326 − 22
E2898 Guide for Risk-Based Validation of Analytical Meth- The rate of this change will depend on the equipment
ods for PAT Applications characteristics, residence volume, and the residence time
E2968 Guide for Application of Continuous Manufacturing distribution/degree of mixing. A fully back-mixed process may
(CM) in the Pharmaceutical Industry be considered and modeled as one or more continuously stirred
E3042 Practice for Process Step to Inactivate Rodent Retro- tank reactors (CSTR). The process to produce a biologic
virus with Triton X-100 Treatment product may contain different modes of manufacturing steps
E3051 Guide for Specification, Design, Verification, and such as batch processing and semi-continuous and continuous
Application of Single-Use Systems in Pharmaceutical and processing steps. For example, the process to make product can
Biopharmaceutical Manufacturing be based on a batch process in a seed expansion stage followed
E3077 Guide for Raw Material eData Transfer from Material by a continuous process in which the cells are growing
Suppliers to Pharmaceutical & Biopharmaceutical Manu- continually in the bioreactor while media and nutrients are
facturers pumped into the vessel and cells and product are removed from
E3231 Guide for Cell Culture Growth Assessment of Single- the bioreactor as an upstream process. The downstream sepa-
Use Material ration of the cells from the media, and each of the subsequent
E3244 Practice for Integrity Assurance and Testing of purification steps, may be run in batch mode or in a semi-
Single-Use Systems continuous or continuous mode based on different manufactur-
ing technologies. If the subsequent steps of the continuous
2.2 ISO Standard:
process step are run in a batch mode as multiple lots, then the
ISO 20399 Biotechnology—Ancillary materials present dur-
product from the continuous process is collected over time.
ing the production of cellular therapeutic products and
This material may be concentrated or is fed to the subsequent
gene therapy products
batch step within predefined ranges for collection, mixing, and
2.3 Regulatory Documents:
hold conditions that assure the solution’s stability over the time
EMA/CHMP/BWP/187338/2014 Guideline on process vali-
the material is collected.
dation for the manufacture of biotechnology-derived ac-
tive substances and data to be provided in the regulatory
3.2.2 batch (or lot), n—specific quantity of material pro-
submission April 28 2016
duced in a process or series of processes that is expected to be
FDA Guidance for Industry PAT A Framework for Innova-
homogeneous within specified limits.
tive Pharmaceutical Development, Manufacturing, and
3.2.2.1 Discussion—In continuous manufacturing (CM), a
Quality Assurance
batch may correspond to a defined fraction of the production
FDA Guidance for Industry Process Validation: General
for either a fixed quantity of material or by the amount
Practices and Principles, rev1
produced in a fixed time interval at constant flow rate. Note
FDA Quality Considerations for Continuous Manufacturing
that multiple lots of raw materials may be used during a CM
Guidance for Industry
process, and it is important to ensure traceability to source in
the event of an excursion at any point.
3. Terminology
3.2.3 dynamic process control system, n—process dynamics
3.1 Definitions—For general definitions, refer to Terminol-
refer to the response of a manufacturing process to changing
ogy E2363 and Guides E2537 and E2475. For definitions
conditions or transient events.
specific to continuous manufacturing, refer to Guide E2968
and ICH Q13 (1). In 3.2, clarification of how they are applied
3.2.3.1 Discussion—An automated control system is one
to bioprocesses is provided.
that (1) monitors the condition of the product or the process or
3.2 Definitions of Terms Specific to This Standard: both, (2) predicts or detects a change to the process indicators
3.2.1 back-mixed process, n—process with a residence time or product quality away from a target condition, and (3) then
distribution (RTD) whose breadth is potentially significant changes the process conditions during manufacturing to main-
compared to the mean residence time. tain the product quality at the target value (or within the
3.2.1.1 Discussion—Certain steps in biomanufacturing are specified range of target values). An example in a continuous
fully back mixed (for example, in the case of a bioreactor or for process producing a biologic would be controlling the cell
a pooled load to a subsequent step) and quantities of material density within a specific range in a perfusion bioreactor by
will be mixed into a single homogeneous condition such that a continuous cell removal (“cell bleed”). Maintaining the density
within that range provides for a higher assurance that the
rapid step change in the properties of inlet material will not
result in an equivalent step change in the properties of the growth rate is constant, the productivity is similar, and the
material produced has the desired characteristics. Depending
output material but will be reflected as a more gradual change.
on the dynamics of the process step, the corrections may be
applied immediately as a step change or as a time-dependent
Available from American National Standards Institute (ANSI), 25 W. 43rd St.,
function (for example, a ramp or exponential function). Such
4th Floor, New York, NY 10036, http://www.ansi.org.
4 real-time control systems may include feedback, feed forward
Available from the European Medicines Agency (EMA), Domenico
Scarlattilaan6, 1083 Amsterdam, The Netherlands, www.ema.europa.eu. control, or 3.2.3.2.
Available from U.S. Food and Drug Administration (FDA), 10903 New
3.2.3.2 multivariate model-based control, n—measurements
Hampshire Ave., Silver Spring, MD 20993, http://www.fda.gov.
of one or more product attributes and process conditions are
The boldface numbers in parentheses refer to the list of references at the end of
this standard. used in a mathematical model of the process or process step to
E3326 − 22
determine the process conditions required to achieve the (fouling) and degradation. Minor changes in process attributes
desired outcome depending on the operational objective (for during the lifespan of a CM operation are acceptable as long as
they remain within preestablished acceptance criteria.
example, cell viability, product titer, and purity) and process
(5) A process consisting of a series of interconnected unit
parameters are adjusted as needed based on the output from the
operations or transformations can be considered to be continu-
model (that is, dynamic control element). It aligns with
ous even if it also contains transformations of defined quanti-
multivariate statistical process control, the application of mul-
ties of material that might be considered to be composed of a
tivariate statistical techniques to analyze complex process data
sequence of discrete events. An example of this is a continuous
with potentially correlated variables. Note that univariate
column purification process in which multiple columns run
controls are also valid.
simultaneously, but each may be at a different stage (for
3.2.4 continuous manufacturing (CM) or manufacturing
example, loading product, washing, elution, regeneration and
step/unit operation, n—involves the continuous feeding of
cleaning), independently of the other columns, such that a
input materials into, the transformation of in-process materials
continuous or semi-continuous flow into the column purifica-
within, and the concomitant removal of output materials from
tion step and out of the column purification step may be
a manufacturing process or unit operation. enabled.
(6) During periods of startup, shutdown, or processing of
3.2.4.1 Discussion—
small quantities of material or both (for example, for
(1) In a CM process or process step, the degree of
development/experimental or clinical studies), it is possible
transformation of any specific quantity of material from an
that not all unit operations within a continuous production line
initial condition into the subsequent condition is a function of
will be in normal or steady state conditions at the same time
the process parameters applied and either:
(for example, startup and shutdown of production cell culture).
(a) The position of the material as it flows through the
This condition should not automatically invalidate the defini-
process,
tion of the process as representative of normal continuous
(b) The duration that the material has been within the
operation.
process, or
3.2.5 process control setpoint, n—specific target value for a
(c) A combination of both (a) and (b).
process parameter or product attribute that is used by a
(2) A CM process or process step may be operated to
dynamic control system.
transform a predefined quantity of material into a product with
3.2.5.1 Discussion—The dynamic process control system
predefined quality attributes that is then subjected to either a
will determine what corrective control action to apply to bring
disposition decision or a decision of the suitability based on the
the specific parameter or attribute closer to the setpoint value.
characteristics of the in-process material. The size of the
A setpoint may be specified together with upper and lower
resulting batch can be defined in terms of one of the following:
target values such that corrective control action may be
(a) Quantity of output material,
reduced once the value is within the specified range. A target
(b) Quantity of input material, and
range specified by upper and lower target values only has no
(c) Run time at a defined mass flow rate.
explicit specified setpoint value and, hence, corrective process
(3) Other approaches to define batch size can also be
control action is often suspended once the parameter or
considered, if scientifically justified based on the characteris-
attribute is within the target range.
tics of the CM process. A batch size can also be defined as a
3.2.6 process disturbance, n—unplanned change to process
range, for example, by defining the minimum and maximum
inputs beyond the normal operating range or conditions (for
run time.
example, process parameter, material property, equipment
(4) A CM process may be operated for an extended time.
condition, or environment) that are introduced into a system.
The quality and quantities of intermediate or finished product
are defined during the operation of the process in a flexible way
3.2.7 process time constant, n—measure of the rate at which
based on principles of science and risk (for example, as any the process can change from steady state operation at one
entity produced in a certain time or containing a certain lot of condition to steady state operation at another condition.
a starting material) and subjected to a disposition decision.
3.2.8 recipe-based process control system, n—automated
Note that in the case of bioprocesses, performance should be
control system that maintains specific process parameters at
monitored to ensure that there are no changes over time. For
prespecified fixed values (that is, according to a predetermined
example, cell-line expression level or product characteristics
recipe) without adjustment of process parameters based on
may change as the cell line ages or if the cell line is
either measurement and feedback of product quality attributes
contaminated with another organism, such as mycoplasma or a
or measurement and feed forward of input material quality
virus. In the case of a purification step, the process parameters
attributes or upstream conditions.
for example resin or membranes used should be monitored to
3.2.9 residence time, n—time that process material is in a
detect changes that may impact product quality attributes or
specific process environment/vessel/unit operation.
yield of the continuous process. For example, the lifetime of a
3.2.10 steady state, n—stable condition that does not change
resin or membrane should be determined using data or labo-
over time.
ratory studies or both and an appropriate cleaning or replace-
ment schedule or both developed to address buildup of material 3.2.10.1 Discussion—
E3326 − 22
(1) Steady state implies that the process is not subject to ensures stability and sterility and provides a dosage form
significant variance with respect to time. consistent with the desired product profile. Operations may
(2) Achieving or maintaining acceptable product quality also include inclusion in a delivery system as a combination
may require an adjustment of target values and, hence, a product. In this guide, operations up to and including final bulk
transition between two steady state conditions. fill for final drugs substance are addressed. Fill/finish opera-
(3) Process parameters can vary within a specified range in tions for drug product and combination products are out of
a steady state process. While critical parameters shall remain scope for this guide.
within a specified tolerance, other parameters may exhibit
4.3 This guide does not advocate the following:
typical process variability.
4.3.1 CM is suitable for the manufacture of all biopharma-
3.2.11 transient event, n—temporary condition in which a
ceutical products and processes;
process goes through a dynamic change.
4.3.2 Guidance on issues related to the safe operation of a
3.2.11.1 Discussion—This change may be due to a distur-
CM process or continuous biomanufacturing equipment. It is
bance or an intentional alteration in the selected operating
the responsibility of the user of this guide to establish appro-
conditions (for example, startup, shutdown, or changes from
priate health and safety practices and determine the applicabil-
one operating condition to another). Development of a con-
ity of regulatory limitations before use; and
tinuous process should include mitigations to prevent process-
4.3.3 Specific designs or operating regimes for CM.
related transient conditions from unacceptably affecting prod-
uct quality. Examples that can result in transient conditions are
5. Operation of Continuous Manufacturing Systems
changes in raw material batches, fouling, a temperature shift in
a cell culture process, or a change in the product quality
5.1 Operational Considerations:
attributes from one process step to another over time. Mitiga-
5.1.1 To introduce CM successfully, due consideration
tions could include diversion to quarantine while the impact of
should first be given to the overall operation and support of the
the conditions on product quality is assessed.
system during the lifecycle of the plant and product, for
example:
4. Significance and Use
5.1.1.1 Considerations for Process and Product
4.1 This guide focuses on upstream and downstream pro-
Development—Use qualified scale-down models of the con-
cesses for biopharmaceutical products with a particular focus
tinuous process or process step to understand the variables that
on antibody production processes. For further information, see
need to be controlled to produce a consistent product and small
Appendix X1 and Refs (1-3).
quantities of material under different operating conditions
during the development of the product. Understanding the
4.2 Bioprocesses traditionally consist of discrete unit opera-
effect of process variables on product properties provides a
tions labeled as upstream, downstream, and fill/finish opera-
basis for designing large-scale manufacturing processes.
tions. The objectives at each stage are significantly different, as
(1) Within the qualified models, consider the uncontrolled
are the operating parameters and control processes, that can
variables that may change over time, for example, changes in
make complete integration impractical initially (Appendix X1).
the genetic makeup of the cells or changes in the proteins
This guide does not imply that complete integration is a
expressed by the cells over time and how that may impact
prerequisite. A higher degree of integration may be possible
product quality. A secondary consideration is the ability to
over time as a better understanding of the dynamics of
detect and characterize process perturbations such as a con-
processes become established.
tamination event.
4.2.1 Upstream Processes—The purpose of upstream pro-
(2) Consider the suitability of the model for manufacture of
cesses is to generate sufficient product to meet patient require-
variable quantities of product at stable operating conditions for
ments preferably in the fewest number of batches. This starts
supplying the product to clinical trials supplies, if applicable.
with increasing biomass (cell-line expansion from working cell
bank to production inoculation) to a production bioreactor in 5.1.1.2 For increasing process capacity from development
which the focus shifts to producing product. The material to commercial production, consider:
within a bioreactor during extended growth is heterogenous, (1) Increasing production by increasing run length
for example, cells will differ in age, there may be genetic drift, duration/number of cycles provided there has been a prior
secreted product can differ in the residence time spent in the verification on the permissible maximum duration of a unit
bioreactor, and cell debris accumulates throughout the process. operation [for example, cell culture, see 5.1.1.1(1)];
4.2.2 Downstream Processes—The purpose of downstream (2) Increasing production by addition of parallel processing
processes is to harvest product and purify it from process- and lines;
product-related impurities (for example, cell debris, nucleic (3) Increase in production rate;
acids, and misfolds) to the desired level. Solids are first (4) A risk-based approach to increasing the scale of CM
separated from solutes; solutes are then separated from each process equipment;
other in the process of purification. Certain processes may at (5) Which parameters can be appropriately characterized
best be semi-continuous, and some steps may be prone to using a scale-down model (in smaller equipment) and which
fouling, which may require manual intervention. parameters are not effectively replicated by the model; and
4.2.3 Fill/Finish Operations—The purpose of fill/finish op- (6) Responding to decreased demand in the same equip-
erations is to formulate the purified product in a form that ment by decreasing batch size/run duration/number of cycles.
E3326 − 22
Note that the change in duration/number of cycles can have an 5.2.2.3 Normal steady state as defined for a particular
impact on validation for the reasons stated previously. process step and in-specification operation (that is, verified to
deliver material that is suitable to be released or move to a
5.1.1.3 For stable manufacturing operations over the target
subsequent process step). As mentioned during this steady state
run length, consider:
operation, specific process characteristics of the cell line or the
(1) The ability of the system to produce consistent product
state of the equipment can be checked such that these variables
over the intended duration of the operation;
remain within acceptable ranges or limits;
(2) Key parameters, sampling points, and process limits
5.2.2.4 Transient operation during flow rate, unit operation
that assure that the process is in a state of control;
changes, or maintenance (for example, replacement of filters as
(3) Mechanisms of failure and degradation of performance
they foul or removal of product/cells at various points to
together with appropriate methods of detection as this detection
maintain cell density levels);
will likely be dependent on the process step being monitored;
5.2.2.5 Replenishment of feedstock materials, particularly
(4) Qualification of recovery procedures in the event of
considering the impact of any variability in raw materials and
excursions (minor nonconformance to catastrophic failure);
processing aids such as media, media feeds, buffers, and filter
(5) Degree of redundancy in equipment and sensors re-
characteristics;
quired to assure continuous stable operation;
5.2.2.6 Process pause or hold or diversion to a surge vessel
(6) Necessity and frequency for operator intervention to
(for example, as a result of alarm conditions);
maintain normal operation (for example, filter fouling); and
5.2.2.7 Process shutdown (including extracting product that
(7) Run-to-run variability in process parameters.
meets specification);
5.1.1.4 In addition, where a site has not previously operated
5.2.2.8 Emptying of equipment or rejection of any residual
a continuous process, consideration should also be given to:
material that does not or would not meet specifications;
(1) Training of development, manufacturing, and quality
5.2.2.9 Cleaning/sanitization/product/grade changeover;
assurance (QA) personnel in the theoretical and practical
5.2.2.10 Controlled safe status (software-controlled safe
aspects of continuous manufacturing;
status (SSS) and hardware-controlled safe status (HSS)); and
(2) Impact of continuous operation on facilities, staff, and
5.2.2.11 Mechanically shut down and out of service.
systems (for example, extended shift working patterns and
5.2.3 In some defined circumstances, manufacturers of drug
deviation management);
substance may reprocess or continue to process material held
(3) Use of equipment that is specifically designed for
under quarantine provided the requirements for rework/reclaim
continuous manufacturing, was adapted for continuous
of the production material are defined in a written procedure
manufacturing, or other technologies such as single-use (SU)
and rework/reclaim is approved by the quality authority to
equipment integrated into the manufacturing process. Most
avoid supply disruption. European guidelines (EMA/CHMP/
(but not all) SU equipment can be supplied presterilized and
BWP/187338/2014) state that reprocessing can be considered
avoid the need for cleaning, in addition to operating as a closed
in exceptional circumstances and when there is a clear identi-
system, which provides a high degree of assurance of sterility.
fication of the root cause. Requirements are no less stringent
See also Guide E3051.
than for traditional batch processes. Examples could include
(4) Appropriate procedure systems to account for real-time
reprocessing of material in the event of a leak or filter failure
or near-real-time rapid release of process intermediates (note
or from a chromatography column in which performance
that hold times may be included/optional as breakpoints to
deviates from criteria (for example, within specification, but
manage the risk of product loss from an excursion further
exceeding action limits). PDA Technical Report 74 provides a
downstream).
detailed discussion of examples and both proactive and reac-
5.2 Operating States:
tive approaches to reprocessing (2). Catastrophic failures (for
5.2.1 The operation of a CM process system shall be
example, gross leaks) are not included.
considered over the entire life cycle of the product (that is,
5.3 Process Management:
development, validation, clinical trial supply, technology
5.3.1 Processes can be sensitive to variability in materials,
transfer, commercial manufacturing, and until product discon-
changes to the process, and/or equipment over time. A fully
tinuation) for which it is intended to be used.
continuous biomanufacturing process (BioCM) or individual
5.2.1.1 A process may begin as a series of connected steps
continual process steps may pose particular challenges because
that are integrated progressively subject to appropriate regula-
of behaviors of both equipment and materials that may be
tory approval before becoming fully continuous.
gradual or stochastic (for example, changes in gene expression
5.2.2 Risk analysis techniques, practical tests, modeling
or cell diameter) and the extended length of the run time. These
tools, or any appropriate combination of these should be used
changes may not be easily observed during batch processing,
to ensure that all potential impacts on product quality are
scaled-down models, or short test runs of continuous processes.
understood and appropriately managed over all potential oper-
5.3.2 Suitable risk analysis, practical tests in-process testing
ating states. For example, the following can be considered:
strategies, and modeling techniques should be considered to
5.2.2.1 Equipment startup (for example, initialization and
determine and evaluate potential challenges in maintaining
warmup ready for manufacturing);
stable process conditions during the operation of a continuous
5.2.2.2 Manufacturing startup (introduction of feed materi- process or a continuous process step over the full length of the
als to start manufacturing and reaching a steady state); required production run, and any sampling or data review as
E3326 − 22
part of this risk analysis and ongoing risk management can be (4) Light exposure (particularly plastic, single use equip-
done in consideration of ongoing process dynamics. ment).
5.3.3.8 There can be changes in plant and equipment char-
5.3.3 Consideration should be given to:
acteristics over time and with prolonged uninterrupted use, for
5.3.3.1 Management of bioburden and the avoidance of
example:
contamination events, which in worst-case scenarios can result
(1) Changes in surface finish and variability in cleaning of
in catastrophic failure and total plant contamination. These are
surfaces;
not unique to continuous manufacturing but the combination of
(2) Changes in clearances because of wear;
extended run times, longer processing times between
(3) Loss of sterility because of wear or improper cleaning
equipment/flow path cleaning and sanitization, and increased
methods; and
process complexity can increase the risk of an adverse event.
(4) Creation of leaks in vessels, tubing, and connectors not
5.3.3.2 Periodic cleaning and sanitization cycles should be
intended for multiple use.
qualified, designed into processes, and validated for effective-
5.3.4 The maximum length of run time of a process depends
ness when the same equipment is used multiple times, for
on the most critical component/step of the process that is
example, in a hybrid process in which multi-use equipment is
susceptible to the extended length of operation and can impact
used together with single-use components. Cleaning cycles are
the product significantly compared to other components or the
included in chromatographic processes to prevent fouling.
steps within the process.
Sanitization steps may be necessary depending on the way
5.3.5 When a single-unit operation within a process line is
columns were prepared for use.
determined to be disproportionally vulnerable to degradation in
5.3.3.3 The potential for fouling and the creation of process
performance or sensitivity to variability, then strategies to
impurities, for example:
maximize the potential run time to avoid the need to stop the
(1) Fouling of equipment surfaces (for example, impact on
overall process can be considered, for example:
heat transfer by product binding);
5.3.5.1 Periodic/rapid replacement of individual items of
(2) Potential impact of binding to surfaces (for example,
equipment such as filters and membranes;
resins, filters) causing product and process modification over
5.3.5.2 Redundancy (particularly for cell retention devices
time;
and membranes used in membrane-based steps),
(3) Ducts, pipes, and tubing changes over time (for
parallelization, or duplication of critical equipment elements
example, impact on flow patterns);
(for example, cell concentration devices, filters, pumps, tubing,
(4) Fouling of instruments and probes (for example, impact
and critical in-process instruments used to measure process
on their accuracy and so forth);
step control parameters); and
(5) Fouling of filters and resins (for example, impact on
5.3.5.3 Process characterization to determine the degree and
flow and pressure of fluids);
frequency of vulnerability of the unit operation and require-
(6) Byproducts with different or undesirable characteristics
ments for preventive maintenance.
or both;
(7) Precipitation, aggregation, encrustation, and/or block- 5.4 Requirement for Operator Intervention:
ing of constrained flow paths; 5.4.1 Generally, a CM can be expected to operate with the
(8) Leachables that could enter the manufacturing process minimum practical level of operator intervention.
during normal operation; and 5.4.2 Certain types of intervention can be planned and a
(9) Creation of aggregates as a consequence of processing course of action determined in the event of, for example, leaks,
steps. filters, or resins fouling.
5.4.3 Unplanned operator intervention should be considered
5.3.3.4 Note it should be accounted that the change out of a
as a potential source of uncontrolled variability. Continued
filter or resin can result in fluctuations in process streams
unplanned intervention may indicate inadequacies in process
(improved flow, increases in product concentration).
design, lack of understanding of the critical process variables,
5.3.3.5 Changes in raw material behavior should be ac-
or uncontrolled or unmanaged variability in process conditions
counted between batches/sources/suppliers that may not be
or raw material properties.
covered within existing quality control requirements, for ex-
5.4.4 Continuous improvement tools (for example, real-time
ample:
statistical process control and process automation) can be used
(1) Biochemical properties of materials and level of
during operation to identify the causes of any unplanned
impurities,
operator intervention. Appropriate actions should be taken to
(2) Electrostatic properties particularly in dried (lyo-
ensure that any impact on product quality is fully understood
philized) biopharmaceuticals,
and that the root cause of the need for intervention is
(3) Safety properties.
eliminated.
5.3.3.6 The impact may be on product quality or process
performance or both.
6. Process Design in a CM Process
5.3.3.7 There can be an impact of environmental changes on
raw material and product, for example: 6.1 Principles:
(1) Temperature, 6.1.1 The design of a CM process step or process requires
(2) Relative humidity (RH), the same good process design and engineering practices used in
(3) Age of raw material, and a traditional batch process.
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6.1.2 However, the design of the CM process may require the bioreactor or purification step. The focus is the time
the consideration of additional factors that are not as important required to produce material in the desired final conditions.
in a batch process, such as fouling potential of cell retention
6.2.2 As the material flows through a particular CM system
devices, product pooling, and fault recovery strategies (a or step, rate-limiting elements within the process shall be
manufacturer may choose to include specific pooling steps at
considered to ensure that, for a given flow rate, the required
strategic points in the process as a precautionary measure to process end point or product attribute can be achieved within
avoid/minimize loss of material in the event of an issue). the time available. For example, the following should be
6.1.3 Hence, when designing a continuous biomanufactur- considered:
ing system, consideration should be given to the process 6.2.2.1 Product binding and elution in a continuous purifi-
conditions experienced by the materials as they flow through cation step,
the system. For example:
6.2.2.2 Ensuring a minimum hold time at a specified con-
6.1.3.1 The overall flow rate through the process (that is, the dition for a viral inactivation step, and
target plant production rate); 6.2.2.3 The time for startup and transient conditions for a
6.1.3.2 The balance between the process and buffer capaci- continuous cell culture process.
ties of each unit operation to ensure that the desired process 6.2.3 An understanding and subsequent verification of the
conditions and overall line flow rates under the required various time constants of the process is specifically important
operating regimes can be achieved. Some examples are: in determining the expected behavior of the process during
(1) Balancing the inflow rate of media into the bioreactor, startup and shutdown and, hence, the impact on quality
with the outflow of product, and drawing off of cells to decisions regarding the disposition of material manufactured
maintain a consistent cell density in the bioreactor growing at during this period.
an appropriate doubling time;
6.2.4 Consideration should be given to the use of monitor-
(2) The capacity of a cell removal system can be examined
ing systems that determine if the required product attributes or
to ensure that it corresponds to the volume and flow rate of the
process indicators are achieved before product is allowed to
cell suspension entering into the cell removal step; and
proceed to the next unit operation.
(3) The rate, flow, and concentration of an unpurified
6.3 Residence Time, Residence Time Distribution, and the
protein solution entering into a continuous purification step can
Degree of Back Mixing:
be managed throughout the individual cycles within the step.
6.3.1 To characterize a continuous process, the process
The flow rate of non-loading steps (for example, cleaning,
residence time and residence time distribution within the CM
regeneration) may be modified to balance with the duration of
step shall be understood and quantified during both startup and
loading steps. This will be dependent on load concentration,
normal operation as well as during process disturbance and
load flow rate, resin dynamic binding capacity, column
shutdown conditions. This is particularly important when the
volume, and the number of columns;
step is dynamically changing, for example, drug substance
6.1.3.3 The instantaneous/peak flow rate at locations in the
production within a continuous cell culture bioreactor.
system where material flow may be discrete;
6.3.2 The mixing of product within the system is important.
6.1.3.4 The flow pattern of the materials in the system (for
For each continuous step, the following should be considered:
example, plug flow versus back mixed);
6.3.2.1 Process modeling of each continuous step and inte-
6.1.3.5 The process conditions required to achieve a specific
gration with prior and following steps (as appropriate),
productivity within the bioreactor or specific purity within a
6.3.2.2 Validation tests using specific markers/tracers, and
continuous purification system;
6.3.2.3 Online/inline process measurement of appropriate
6.1.3.6 The process time constants, reaction rates, average,
process indicators and product attributes.
maximum, and minimum residence times required to achieve a
6.3.3 Two extremes of mixing are commonly identified as
specific process objective such as specific product quality
“plug flow” or “fully back mixed.” Bioreactor processes have
(glycosylation, recovery, purity);
a high degree of mixing. Product that has been produced by
6.1.3.7 The relationship between material properties, pro-
cells in the bioreactor, but not yet removed, may be degraded
cess conditions, and equipment design required to achieve a
by host cell impurities that are retained in the bioreactor.
reliable flow of materials;
6.3.4 An estimation of the residence time distribution (RTD)
6.1.3.8 The analysis of the mass and energy balance for the
within the process enables an understanding of:
system or continuous process step using process and chemical
6.3.4.1 Which output material contains which input
engineering principles, for example:
material,
(1) Capacity of physical transfer systems,
6.3.4.2 Which process conditions have had an impact on a
(2) Capacity of heating systems, and
specific quantity of output material,
(3) Sufficient supply of nutrients and removal of by-
6.3.4.3 How minor and transient changes in feed or process
products for a growing cell culture; and
conditions will impact output product attributes, and
6.1.3.9 Implementation of appropriate monitoring tools.
6.3.4.4 The degree of recycle.
6.2 Process Time Constants:
6.3.5 In particular, quantification of the residence time
6.2.1 The time available for a given bioproduct production distribution may be used to ensure that product remains within
or purification is determined by the residence time of the the predefined process conditions. This understanding is par-
material in a specific process step based on the environment in ticularly important in live cultures of cells in which strict
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control is necessary to ensure the viability and or properties of opportunity to gather more information that will aid in process
the live cells and their product. Similarly, continuous purifica- characterization and further advancing process understanding.
tion steps require control of some variables (pH, temperature)
7.5 Sampling and Data Collection within a CM Process:
that could cause major changes in the properties of the product.
7.5.1 The timing and appropriateness of measurements used
6.3.6 Understanding and quantification of the residence
to monitor and control a continuous process or process step
time distribution is also important when the product impact of
will depend on the purpose and the nature of the step (for
deviations from process parameters need to be assessed and
example, product titer in a bioreactor versus impurity profile
product disposition decisions need to be made.
further downstream) as well as the following characteristics:
6.4 Product Transport and Material Properties:
7.5.1.1 The intended scale of scrutiny of the sampling and
6.4.1 A CM process frequently consists of a number of
measurement system;
connected unit operations.
7.5.1.2 The impact of product flow on product characteris-
6.4.2 Careful consideration should be given to the design of
tics;
transport and flow control elements within a continuous system
7.5.1.3 The extent that measurement made is representative
to ensure that materials will flow in a predictable way without
of the complete process stream; and
adverse impact on product quality.
7.5.1.4 The impact of process dynamics on the requirement
6.4.3 Transporting and controlling the flow rate of solutions
of the sampling and measurement system, which may be
or suspension of cells may be a specific problem in this respect.
limiting on the flow rate at which a continuous unit operation
For example, rapid transport of cells to and from the cell
can be run. The impact of parameters that may vary within that
retention device may cause cell lysis; excessive agitation may
process step on quality attributes or process performance or
cause aggregation of product because of excessive air\liquid
both should be understood, so that the relationship between
interface contact. Hence, the handling and flow properties of
changes, measurements, and effective response times are har-
materials to be processed should be determined as early as
monized with each other.
possible within the development of the product such that the
7.5.2 The frequency of measurements should be appropriate
process equipment may be designed appropriately.
to the dynamic response time of the parameter or attribute.
6.4.4 Characterization of materials using laboratory tech-
7.5.3 Due consideration should be given to how to handle
niques on small samples may give good early indication of
measurement noise caused by variability in sample
potential problems. But where there are concerns about mate-
presentation, which is potentially greater in an online system
rial properties, it is recommended that
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