E55.12 - Process Applications

SIGNIFICANCE AND USE
3.1 Rodent-derived cell lines are widely used in the production of biopharmaceutical drugs such as mAbs and Fc fusion proteins. These cell lines have been shown to contain genes encoding endogenous retroviral-like particles or endogenous retrovirus. Despite the lack of evidence for an association between such rodent retroviruses and disease in humans, the potential contamination of human therapeutics raises safety concerns for biopharmaceutical drugs. Additionally, adventitious agents such as viruses can be introduced into a biopharmaceutical drug substance manufacturing process from other sources, and potential safety issues can be attributed to these potential unknowns. For these reasons, effective viral clearance is an essential aspect of an integrated approach combining safety testing and process characterization which ensures virus safety for biopharmaceutical drug products made using rodent cell lines.  
3.2 Solvent/detergent inactivation has been widely used for decades to inactivate enveloped viruses in blood plasma derived biopharmaceutical therapies (1-3).3 Solvent/detergent systems using the detergents Triton X-100 or Polysorbate 80 along with the organic solvent tri(n-butyl)phosphate (TNBP) have been used to inactivate enveloped viruses by disrupting the viral envelope thereby reducing the ability of the enveloped virus to attach to and then infect the host cell (4 and 5).  
3.3 Most manufacturers of mAbs, recombinant proteins, and Fc fusion proteins have focused on viral inactivation methods using the detergent Triton X-100 or Polysorbate 80 in the absence of TNBP (6), which can interfere with subsequent bioprocessing steps. The ability of the detergents alone to inactivate retroviruses has been demonstrated in monoclonal antibodies produced in rodent-derived cell lines (6-9). At a 2011 workshop devoted to viral clearance steps used in bioprocessing (7), investigators from one firm showed incubation with 0.2 % Triton X-100 for 60 min of hold time at a...
SCOPE
1.1 This practice assures effective inactivation of ≥4 log10 of infectious rodent retrovirus (that is, reduction from 10 000 to 1 infectious rodent retrovirus or removal of 99.99 % of infectious rodent retroviruses) in the manufacturing processes of monoclonal antibodies or immunoglobulin G (IgG) Fc fusion proteins manufactured in rodent-derived cell lines that do not target retroviral antigens. Rodent retrovirus is used as a model for rodent cell substrate endogenous retrovirus-like particles potentially present in the production stream of these proteins.  
1.2 The parameters specified for this practice are clarification, Triton X-100 detergent concentration, hold time, pH, and inactivation temperature.  
1.3 This practice can be used in conjunction with other clearance or inactivation unit operations that are orthogonal to this inactivation mechanism to achieve sufficient total process clearance or inactivation of rodent retrovirus.  
1.4 This detergent inactivation step is performed on a clarified, cell-free intermediate of the monoclonal antibody or IgG Fc fusion protein.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 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.

SIGNIFICANCE AND USE
3.1 Mammalian cell lines are widely used in the production of biological therapeutics, such as monoclonal antibodies and other recombinant proteins. Some of these cell lines, like rodent cell lines, are known to contain genes encoding endogenous retroviral-like particles or produce endogenous retrovirus, but there is no evidence of an association between rodent retrovirus and disease in humans. Adventitious viruses can be introduced into a drug substance manufacturing process from other sources, and contamination of human therapeutics is a safety concern (3).  
3.2 Virus filtration, an orthogonal technology in a virus clearance platform to such steps as low pH or surfactant inactivation, has traditionally been accepted as a robust method for virus clearance when well designed. Size exclusion has been shown to be the primary mechanism of virus removal by virus retentive filtration, that is, larger viruses are more easily retained than smaller viruses such as parvoviruses (4, 5). Large virus retention has also been shown to be insensitive to process fluid characteristics such as protein type, protein concentration, pH, and ionic strength (4, 6, 7, 8, 9, 10). In contrast, for small viruses, aspects like flow pausing and/or flux decay can impact clearance (4, 6, 11).  
3.3 Large virus retentive filters, or retrovirus filters, are tested for removal of larger enveloped viruses like retrovirus or MuLV (80 nm to 100 nm) and have undetectable levels of the large bacteriophage PR772 (64 nm to 82 nm) (1). Small virus retentive filters, or parvovirus filters, are designed to remove parvovirus, like MMV (18 nm to 26 nm) (1). Since size exclusion has been demonstrated as the mechanism of virus retention, retroviruses, which are three to four times larger than parvoviruses, should be large enough to be completely retained, with undetectable levels of retrovirus in the filtrate, by all small virus retentive filters designed to remove parvovirus.  
3.4 Numerous published studies...
SCOPE
1.1 This practice assures 6.0 log10 removal of retrovirus (for example, MuLV).  
1.2 This practice is applicable to monoclonal antibody (mAb), immunoglobulin G (IgG) fusion proteins, recombinant proteins, or other proteins produced using mammalian cell lines (for example, Chinese hamster ovary (CHO), murine hybridomas, murine myelomas, or human embryonic kidney (HEK) 293).  
1.3 The step is performed on cell-free intermediates.  
1.4 The log removal claim for retrovirus by small virus retentive filters can be used in conjunction with other clearance unit operations (for example, low pH inactivation, or inactivation of virus by surfactant) to assure sufficient total process clearance of potential virus contaminants, which would be supportive of early phase (clinical phase 1 or phase 2a trials) regulatory filings.  
1.5 Retrovirus removal claim by filtration is limited to small virus retentive filters, as defined in the PDA Technical Report Virus Filtration (1)2 in the context of this standard.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.8 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.

SIGNIFICANCE AND USE
4.1 This guide focuses on upstream and downstream processes for biopharmaceutical products with a particular focus on antibody production processes. For further information, see Appendix X1 and Refs (1-3).  
4.2 Bioprocesses traditionally consist of discrete unit operations labeled as upstream, downstream, and fill/finish operations. The objectives at each stage are significantly different, as are the operating parameters and control processes, that can make complete integration impractical initially (Appendix X1). This guide does not imply that complete integration is a prerequisite. A higher degree of integration may be possible over time as a better understanding of the dynamics of processes become established.  
4.2.1 Upstream Processes—The purpose of upstream processes is to generate sufficient product to meet patient requirements preferably in the fewest number of batches. This starts with increasing biomass (cell-line expansion from working cell bank to production inoculation) to a production bioreactor in which the focus shifts to producing product. The material within a bioreactor during extended growth is heterogenous, for example, cells will differ in age, there may be genetic drift, secreted product can differ in the residence time spent in the bioreactor, and cell debris accumulates throughout the process.  
4.2.2 Downstream Processes—The purpose of downstream processes is to harvest product and purify it from process- and product-related impurities (for example, cell debris, nucleic acids, and misfolds) to the desired level. Solids are first separated from solutes; solutes are then separated from each other in the process of purification. Certain processes may at best be semi-continuous, and some steps may be prone to fouling, which may require manual intervention.  
4.2.3 Fill/Finish Operations—The purpose of fill/finish operations is to formulate the purified product in a form that ensures stability and sterility and provides a dosage form consis...
SCOPE
1.1 This guide is intended as a complement to Guide E2968. It provides key concepts and principles to assist in the appropriate selection, development, and operation of continuous processing technologies for the manufacture of biologically derived products.  
1.2 Several of the principles covered in Guide E2968 are applicable to biomanufacturing. However, processes for biologically derived products differ from those for synthetic drugs in a number of fundamental ways in addition to their source (for example, format: aqueous liquids versus powders; scope: genesis to final formulation). This guide is intended to provide greater clarity for biomanufacturing. It does not imply that topics in Guide E2968 that are not covered here do not apply to continuous manufacturing (CM) for biologics.  
1.3 Biologically derived products also differ widely from each other in terms of modalities, source materials, and the manufacturing technologies used, not all of which are equally amenable to operating in a continuous mode.  
1.4 Opportunities do exist for the introduction of continuous technologies, for example, efforts are ongoing to adapt processes for large-scale manufacture of broadly applicable modalities such as monoclonal antibodies to a continuous format. This guide is intended to provide guidance to the design and implementation of antibody processes.  
1.5 The principles can be applicable to unit operations or processes or both for other modalities but may not be applicable to all bioprocesses.  
1.6 Particular consideration should be given to the development and application of the appropriate scientific understanding and engineering principles that differentiate CM from traditional batch manufacturing.  
1.7 Since much of the processing is done under conditions amenable to microbial growth, maintaining process streams free from external biological impurities and microbial contamination (for example, bioburden, viruses, ...

SIGNIFICANCE AND USE
2.1 This practice outlines a procedure for the mechanical calibration of paddle and basket dissolution units to ensure reproducibility of results.  
2.2 Once a unit meets all of the mechanical specifications included in this practice, it is considered calibrated and further calibration with dissolution calibrator tablets is not required.
SCOPE
1.1 This practice covers the set-up and calibration of the paddle and basket dissolution apparatus.  
1.2 Use of this practice may be applied to apparatus that have been modified to enable automatic dissolution testing (that is, a valve in the bottom of the vessel or sampling through the shaft).  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 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.

SCOPE
1.1 This practice assures 5 log10 inactivation of non-defective C-type retroviruses, which are endogenous to murine hybridoma and CHO cells and are potentially present in the production stream of biopharmaceutical processes that use rodent derived cell culture.  
1.2 The process parameters specified in this practice consistently assure 5 log10 inactivation of murine retrovirus by adjusting the pH of a process solution after initial affinity capture chromatography purification.  
1.3 This practice is applicable to mAb, IgG fusion, or other recombinant proteins produced from rodent cell lines (for example, CHO or murine hybridoma), which do not target retroviral proteins. Additionally, the low pH step is performed on a cell-free intermediate, post initial capture using protein A chromatography.  
1.4 The 5 log10 inactivation of murine retrovirus claimed by using this practice will be utilized in conjunction with other clearance unit operations (for example, chromatography and virus retentive filtration) to assure sufficient total process clearance of murine retroviruses, which will be supportive of early phase regulatory filings.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 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.

SIGNIFICANCE AND USE
5.1 Pharmaceutical water is the most common component or ingredient used in pharmaceutical and biopharmaceutical manufacturing. Acceptable purity of the water is important to the quality of the final pharmaceutical product. TOC concentration is a key indicator and attribute of the purity of this water and also an important monitor of the overall performance of the water purification system. TOC analysis is the measurement of all the covalently bound carbon present in the water, not including carbon in the form of carbon dioxide (CO2), bicarbonate icon (HCO3 –), or carbonate ion (CO3 2–), and is reported as the mass of organic carbon per volume.  
5.2 Application of this practice provides pertinent information to make informed decisions on the release of water meeting pharmaceutical TOC concentration specifications.
SCOPE
1.1 This practice establishes an approach to the real-time release testing (RTRT) of pharmaceutical water based on the total organic carbon (TOC) attribute using on-line total organic carbon (OLTOC) instrumentation that is in agreement with current regulatory thinking.  
1.2 This practice is harmonized with or supports the concepts of relevant ASTM International Committee E55 on Manufacture of Pharmaceutical Products standards, ICH Harmonized Tripartite Guidelines, the U.S. FDA PAT Guidance, and U.S. FDA Pharmaceutical cGMPs.  
1.3 This practice does not provide general guidance information for pharmaceutical procedures that are considered standard practice in the pharmaceutical industry. This practice provides specific guidance for non-standardized procedures.  
1.4 This practice does not address the user’s various internal procedures for risk, change, or quality management systems. The overall project effort associated with this practice shall be proportional to the overall risk of failing the pharmaceutical water’s TOC concentration specification.  
1.5 This practice does not purport to establish how to comply with pharmacopeias. The RTRT methodology selected must assure compliance with the user’s current required pharmacopeias. However, compliance with pharmacopeia TOC methods is not necessarily sufficient to meet current regulatory expectations for RTRT.  
1.6 This practice does not purport to substitute for or replace compendial bioburden testing requirements. It is strictly applicable to the TOC attribute of water quality.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
3.1 Rodent-derived cell lines are widely used in the production of biopharmaceutical drugs such as mAbs and Fc fusion proteins. These cell lines have been shown to contain genes encoding endogenous retroviral-like particles or endogenous retrovirus. Despite the lack of evidence for an association between such rodent retroviruses and disease in humans, the potential contamination of human therapeutics raises safety concerns for biopharmaceutical drugs. Additionally, adventitious agents such as viruses can be introduced into a biopharmaceutical drug substance manufacturing process from other sources, and potential safety issues can be attributed to these potential unknowns. For these reasons, effective viral clearance is an essential aspect of an integrated approach combining safety testing and process characterization which ensures virus safety for biopharmaceutical drug products made using rodent cell lines.  
3.2 Solvent/detergent inactivation has been widely used for decades to inactivate enveloped viruses in blood plasma derived biopharmaceutical therapies (1-3).3 Solvent/detergent systems using the detergents Triton X-100 or Polysorbate 80 along with the organic solvent tri(n-butyl)phosphate (TNBP) have been used to inactivate enveloped viruses by disrupting the viral envelope thereby reducing the ability of the enveloped virus to attach to and then infect the host cell (4 and 5).  
3.3 Most manufacturers of mAbs, recombinant proteins, and Fc fusion proteins have focused on viral inactivation methods using the detergent Triton X-100 or Polysorbate 80 in the absence of TNBP (6), which can interfere with subsequent bioprocessing steps. The ability of the detergents alone to inactivate retroviruses has been demonstrated in monoclonal antibodies produced in rodent-derived cell lines (6-9). At a 2011 workshop devoted to viral clearance steps used in bioprocessing (7), investigators from one firm showed incubation with 0.2 % Triton X-100 for 60 min of hold time at a...
SCOPE
1.1 This practice assures effective inactivation of ≥4 log10 of infectious rodent retrovirus (that is, reduction from 10 000 to 1 infectious rodent retrovirus or removal of 99.99 % of infectious rodent retroviruses) in the manufacturing processes of monoclonal antibodies or immunoglobulin G (IgG) Fc fusion proteins manufactured in rodent-derived cell lines that do not target retroviral antigens. Rodent retrovirus is used as a model for rodent cell substrate endogenous retrovirus-like particles potentially present in the production stream of these proteins.  
1.2 The parameters specified for this practice are clarification, Triton X-100 detergent concentration, hold time, pH, and inactivation temperature.  
1.3 This practice can be used in conjunction with other clearance or inactivation unit operations that are orthogonal to this inactivation mechanism to achieve sufficient total process clearance or inactivation of rodent retrovirus.  
1.4 This detergent inactivation step is performed on a clarified, cell-free intermediate of the monoclonal antibody or IgG Fc fusion protein.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.