By Leah Choi
Four grueling years of chemistry, biology, math, and physics barely prepared me for life after college. As I entered America’s workforce on November 13th, 2006, I was equipped with nothing more than a general knowledge of what was to come.
Within my first few weeks at RMC Pharmaceutical Solutions, I quickly realized my inadequacies. GMP? GLP? NDA? IND? The acronyms alone could have driven me to near insanity. Likewise, I had spent four years at the University of Colorado learning the theory behind chromatography even putting it into practice on an ancient gas chromatography system. Yet this was no match for the advanced chromatography systems used in today’s biotech industry. To complicate matters further, I did not fully understand the intricacies of working in a regulated environment. What did it mean to follow a standard operating procedure? To evaluate and qualify the design, installation, performance, and operation of an instrument? What did it mean to document deviations? To perform a corrective and preventative action? Each new client and each new project presented a fresh set of unfamiliar issues. I often questioned if I would ever be able to bridge the gap between the theories of my college education with the applications of my working world.
Am I an isolated incident or do current biotechnology educational programs lack the necessary curriculum to develop entry-level employees? According to a recent survey conducted by AAPS (American Association of Pharmaceutical Sciences) and published by the National Institute of Pharmaceutical Technology and Education (NIPTE), 35% of respondents believe that current training for entry-level pharmaceutical development scientists is inadequate, 60% believe that there is a shortage of suitable candidates and nearly 70% asserted that there is an inadequacy in the number of US colleges focusing on industrial needs. According to this survey, academic programs training the majority of pharmaceutical product development scientists have declined substantially in recent years due to the emphasis in professional pharmacy programs on patient care rather than product knowledge. Additionally, these programs lack research funding in basic physical sciences supporting development and manufacturing.
With so much on the line, what is currently being done to address these nationally recognized problems? Organizations such as NIPTE have implemented plans with hopes of providing the “highest caliber entry-level scientists/engineers for the pharmaceutical and biopharmaceutical industries”. These plans include training students in degree programs using shared curricula materials, summer training programs and industrial internships via a network of industrial and institutional collaborators. The curriculum is based on “the precepts of interdisciplinary approaches strongly advocated by the National Academy of Sciences and constructivist learning theories important in the development of modern engineering and science higher education”. Presently, only ten universities have signed onto this plan: Duquesne University, Illinois Institute of Technology, Purdue University, Rutgers University, University Puerto Rico San Juan/Mayaguez, University of Connecticut, University of Iowa, University of Kansas, University of Kentucky, University of Maryland-Baltimore, and the University of Minnesota.
Other initiatives include the National Science Foundation (NSF) which currently provides $16.3 million in support of biotechnology programs through its Advance Technological Education (ATE) program. NSF regularly brings together scientists, educators, and other stakeholders to share their opinions on official issues such as biotechnology workforce development. Panelists share their opinions about how the biotechnology industry will grow during the next five years, the skills that technicians will require to meet workforce needs, and their experiences with promising educational practices. April 2008 conference recommendations include
1. instruction in written and verbal communication and “soft skill” such as team work and time management;
2. core curriculum courses that transfer and articulate from high school to two-year and four-year degree programs;
3. a strong theoretical understanding of the entire manufacturing process encompassing upstream and downstream process;
4. the introduction of immerging technologies in basic biotechnology courses; and
5. the redesign of standard microbiology and biology curricula to include applications in industrial and environmental biotechnology.
The limitations of ordinary degree programs spurred Montgomery College in Maryland to develop curriculum specifically aimed at preparing students for life after college. By soliciting information from industry personnel, coordinators of the program have developed and continually update courses that meet the current skill sets expected from entry level employees. Students acquire real life experience through internships engineered by the program. The success of this program depends highly on the continual collaboration between working professionals and academic faculty. Without a doubt, this synergistic dynamic allows both sides to benefit: educators gain valuable input for relevant curriculum while industry gains practical, productive entry level employees. Programs like these emphasize the absolute necessity for industry involvement.
I realize the learning curve is steep but not unconquerable. To all those recent graduates or better yet, those who are still in school the best advice I can give, is to get involved. Build your own bridge, by participating in professional groups and societies. Attend local events and seminars. Organizations such as the PDA (Parental Drug Association) AAPS (American Association of Pharmaceutical Scientists) and ISPE (International Society of Professional Engineers) often provide students with special benefits and discount memberships. As a PDA Student Member, you receive access to numerous benefits which provide you with the most current scientific and technical information. You receive access to Student Programs which provide grant funding and career growth resources, subscriptions to the PDA Journal of Pharmaceutical Science and Technology, including PDA Technical Reports which offer expert guidance and opinions on a variety of important scientific and regulatory topics pertaining to pharmaceutical and biopharmaceutical production.
Begin building contacts as early as possible. Make your first professional contacts at a career fair. Collect business cards and follow up. Professional social networking sites such as LinkedIn, Xing, Plaxo, and Spoke make these connections simple and instantaneous. Seek and invite professionals to speak at your school’s student group and find other means of collaborating with industry experts. Build a bond with someone who can act as a mentor. Having a mentor can be a great way to develop your career for the long term.
Take the opportunity to educate yourself on current topics and those of interest. Earlier this year, I was afforded the opportunity to gain valuable knowledge by earning a Certificate of Good Laboratory Practices/Good Manufacturing Practices from the University of Denver. We spent 10 weeks focusing on the regulations surrounding device manufacturing and use, specifically 21CFR820 and 21CFR58. My thirst for knowledge did not end there. In the same months I earned a Yellow Belt Training Certificate from the Colorado Association of Manufacturing and Technology. For six weeks my classmates and I studied yellow belt topics, specifically in the areas of Six Sigma Root Cause Analysis, 8D Problem Solving and Statistical Process Controls. Both of these courses were free, local and most importantly, offered helpful insights into contemporary subjects.
I commend unique programs and organizations such as NIPTE, NSF and Montgomery College for offering much needed curriculum that unites academia with industry. With increasing awareness and initiatives, my hope is that future graduates will be endowed with the basic education and ability to hit the ground running within this rapidly emerging industry.
Thursday, January 21, 2010
Thursday, January 14, 2010
Is Bovine Polyoma Virus Getting You Down?
By Dr. Ray Nims
Bovine polyomavirus (BPyV) is a double-stranded DNA virus of genus Polyomavirus. It is non-enveloped and 40-50 nm in diameter, and is a member of the same genus as SV40.
Basis of Concern. The Polyomavirus genus was so-named due to the ability of the viruses to cause tumors in susceptible host animals. Genomic sequences for the potentially oncogenic bovine polyomavirus have been detected with high frequency in bovine sera, regardless of geographic region of origin (Shuurman et al., J. Gen. Virol. 72: 2739-2745, 1991; Wang et al., New Zealand Vet. J. 53: 26-30, 2005).
Regulatory Expectations. Bovine polyomavirus is not mentioned specifically in 9CFR 113.47, Detection of extraneous viruses by the fluorescent antibody technique, as a virus of concern for raw materials of bovine origin. As a result, BPyV is not specifically probed for during most 9CFR 113.53-based raw material viral infectivity testing, and this test most likely would not be capable of detecting BPyV if present in the test material. The EMEA Note for Guidance on the use of Bovine Serum in the Manufacture of Human Biological Medicinal Products (CPMP/BWP/1793/02) states that sera users are “encouraged to apply infectivity assays for BPyV and to investigate methods for inactivation/removal of BPyV in order to limit or eliminate infectious virus from batches of serum”.
Mitigating Risk. In actual practice, the available infectivity assays for BPyV involve numerous passages using a bovine detector cell such as MDBK and are somewhat lengthy and insensitive, though more sensitive assays are under development. Cell-based infectivity testing for BPyV is not always being performed by users for each batch of bovine serum. The lack of a rapid and sensitive infectivity assay also means that viral inactivation studies for BPyV are not practically possible. While another polyomavirus such as SV40 could be used in viral inactivation/removal studies as a proxy for BPyV, in actual practice the murine parvovirus MMV (mouse minute virus) is more typically used as a worst-case model virus for such studies since it is non-enveloped and even smaller than BPyV. The few studies performed with SV40 indicate that gamma-irradiation at the dosages normally employed is not effective at inactivating this virus, as might be expected for a virus of this relatively small size (e.g., Gauvin, 2009). On the other hand, it has been shown (Wang et al., Vox Sanguinis 86: 230-238, 2004) that UVC treatment is effective in inactivating SV40. Note: since originally authoring this blog, I have come across a great number of UV-inactivation papers which indicate that polyomaviruses, and SV-40 in particular, appear to be relatively resistant to UV inactivation. The Wang et al. result may represent an outlier. I will address this in a future blog. Studies using MMV indicate that high-temperature short-time (HTST)-treatment of medium containing bovine serum is effective in inactivating this virus (Schleh et al., Biotechnol. Prog. 25: 854-860, 2009), and would by implication be effective for BPyV.
Conclusions. At the present time, infectivity screening of bovine sera for BPyV is not always being performed, and it is believed that the high frequency of detection of genomic material in bovine sera may not reflect a similarly high frequency of infectious BPyV. Risk of infection of biological products with BPyV through use of bovine-derived materials such as bovine sera may be mitigated through implementation of UVC- or HTST-treatment of media containing the sera and of viral purification processes capable of removing and inactivating an even smaller non-enveloped virus such as MMV.
Bovine polyomavirus (BPyV) is a double-stranded DNA virus of genus Polyomavirus. It is non-enveloped and 40-50 nm in diameter, and is a member of the same genus as SV40.
Basis of Concern. The Polyomavirus genus was so-named due to the ability of the viruses to cause tumors in susceptible host animals. Genomic sequences for the potentially oncogenic bovine polyomavirus have been detected with high frequency in bovine sera, regardless of geographic region of origin (Shuurman et al., J. Gen. Virol. 72: 2739-2745, 1991; Wang et al., New Zealand Vet. J. 53: 26-30, 2005).
Regulatory Expectations. Bovine polyomavirus is not mentioned specifically in 9CFR 113.47, Detection of extraneous viruses by the fluorescent antibody technique, as a virus of concern for raw materials of bovine origin. As a result, BPyV is not specifically probed for during most 9CFR 113.53-based raw material viral infectivity testing, and this test most likely would not be capable of detecting BPyV if present in the test material. The EMEA Note for Guidance on the use of Bovine Serum in the Manufacture of Human Biological Medicinal Products (CPMP/BWP/1793/02) states that sera users are “encouraged to apply infectivity assays for BPyV and to investigate methods for inactivation/removal of BPyV in order to limit or eliminate infectious virus from batches of serum”.
Mitigating Risk. In actual practice, the available infectivity assays for BPyV involve numerous passages using a bovine detector cell such as MDBK and are somewhat lengthy and insensitive, though more sensitive assays are under development. Cell-based infectivity testing for BPyV is not always being performed by users for each batch of bovine serum. The lack of a rapid and sensitive infectivity assay also means that viral inactivation studies for BPyV are not practically possible. While another polyomavirus such as SV40 could be used in viral inactivation/removal studies as a proxy for BPyV, in actual practice the murine parvovirus MMV (mouse minute virus) is more typically used as a worst-case model virus for such studies since it is non-enveloped and even smaller than BPyV. The few studies performed with SV40 indicate that gamma-irradiation at the dosages normally employed is not effective at inactivating this virus, as might be expected for a virus of this relatively small size (e.g., Gauvin, 2009). On the other hand, it has been shown (Wang et al., Vox Sanguinis 86: 230-238, 2004) that UVC treatment is effective in inactivating SV40. Note: since originally authoring this blog, I have come across a great number of UV-inactivation papers which indicate that polyomaviruses, and SV-40 in particular, appear to be relatively resistant to UV inactivation. The Wang et al. result may represent an outlier. I will address this in a future blog. Studies using MMV indicate that high-temperature short-time (HTST)-treatment of medium containing bovine serum is effective in inactivating this virus (Schleh et al., Biotechnol. Prog. 25: 854-860, 2009), and would by implication be effective for BPyV.
Conclusions. At the present time, infectivity screening of bovine sera for BPyV is not always being performed, and it is believed that the high frequency of detection of genomic material in bovine sera may not reflect a similarly high frequency of infectious BPyV. Risk of infection of biological products with BPyV through use of bovine-derived materials such as bovine sera may be mitigated through implementation of UVC- or HTST-treatment of media containing the sera and of viral purification processes capable of removing and inactivating an even smaller non-enveloped virus such as MMV.
Thursday, January 7, 2010
Quality by Design: Dissolution Time
By Dr. Scott Rudge
In a previous post, I discussed the prevalence of statistics used in Quality by Design. These statistical tools are certainly useful and can provide (within their limits of error) prediction of future effects of excursions from control ranges for operating parameters, specifically for Critical Quality Attributes (CQA’s). The limitations of this approach were discussed in the previous blog. In the next series of blogs on Quality by Design, I will discuss opportunities for increasing quality, consistency and compliance for biotechnology products by building quality from the ground up.
While active pharmaceutical ingredient (API) manufacture by biosynthesis is a complicated and difficult to control prospect, there are a number of fundamental operations that are imminently controllable. Media and buffers must be compounded, sometimes adjusted or supplemented, stored and ultimately used in reactors and separators to produce and purify the API. These solutions are fairly easy to make with precision. Three factors come immediately to mind that can be known in a fashion that is scale independent and rigorous, 1) the dissolution rate, 2) the mixing power required and 3) the chemical stability of the solution.
The dissolution rate is a matter of mass transfer from a saturated solution at the dissolving solid interface to the bulk solution concentration. If particle size is fairly consistent, then the dissolution rate is represented by this equation:
where k is the mass transfer coefficient, provided the dissolving solid is fully suspended. It is easy to measure this mass transfer rate in the laboratory with an appropriate measure of solution concentration. For example, for the dissolution of sodium chloride, conductivity can be used. We conducted such experiments in our lab across a range of volumes salt concentrations, and found a scale independent mass transfer coefficient of approximately 0.4 s-1. An example of the results is shown in the accompanying figure.
With the mass transfer coefficient in hand, the mixing time can be precisely specified, and an appropriately short additional engineering safety factor added. If the times are known for dissolution, and mixing is scaled appropriately (as will be shown in future blogs) then buffers and other solutions can be made with high precision and little wasted labor or material. In addition, the properties of the solutions should be constant within a narrow range, and the reproducibility of more complicated unit operations such as reactors and separators, much improved.
A design based on engineering standards such as this produces predictable results. Predictable results are the basis of process validation. As the boiling point of water drives the design of the WFI still, we should let engineering design equations drive Quality by Design for process unit operations.
Leah Choi contributed to this work
In a previous post, I discussed the prevalence of statistics used in Quality by Design. These statistical tools are certainly useful and can provide (within their limits of error) prediction of future effects of excursions from control ranges for operating parameters, specifically for Critical Quality Attributes (CQA’s). The limitations of this approach were discussed in the previous blog. In the next series of blogs on Quality by Design, I will discuss opportunities for increasing quality, consistency and compliance for biotechnology products by building quality from the ground up.
While active pharmaceutical ingredient (API) manufacture by biosynthesis is a complicated and difficult to control prospect, there are a number of fundamental operations that are imminently controllable. Media and buffers must be compounded, sometimes adjusted or supplemented, stored and ultimately used in reactors and separators to produce and purify the API. These solutions are fairly easy to make with precision. Three factors come immediately to mind that can be known in a fashion that is scale independent and rigorous, 1) the dissolution rate, 2) the mixing power required and 3) the chemical stability of the solution.
The dissolution rate is a matter of mass transfer from a saturated solution at the dissolving solid interface to the bulk solution concentration. If particle size is fairly consistent, then the dissolution rate is represented by this equation:
where k is the mass transfer coefficient, provided the dissolving solid is fully suspended. It is easy to measure this mass transfer rate in the laboratory with an appropriate measure of solution concentration. For example, for the dissolution of sodium chloride, conductivity can be used. We conducted such experiments in our lab across a range of volumes salt concentrations, and found a scale independent mass transfer coefficient of approximately 0.4 s-1. An example of the results is shown in the accompanying figure.
With the mass transfer coefficient in hand, the mixing time can be precisely specified, and an appropriately short additional engineering safety factor added. If the times are known for dissolution, and mixing is scaled appropriately (as will be shown in future blogs) then buffers and other solutions can be made with high precision and little wasted labor or material. In addition, the properties of the solutions should be constant within a narrow range, and the reproducibility of more complicated unit operations such as reactors and separators, much improved.
A design based on engineering standards such as this produces predictable results. Predictable results are the basis of process validation. As the boiling point of water drives the design of the WFI still, we should let engineering design equations drive Quality by Design for process unit operations.
Leah Choi contributed to this work
Wednesday, December 30, 2009
Can Cache Valley Virus Trash Your Manufacturing?
By Dr. Ray Nims
Cache Valley virus is a single-stranded RNA virus of family bunyavirus, genus Bunyavirus. It is enveloped and nominally 80-120 nm in diameter. Cache Valley virus was first isolated in Utah in 1956 and is carried by mosquitoes. It has since been found to be a widespread virus, having been isolated in Texas, Michigan, North Carolina, Indiana, Virginia and Maryland, for example.
Basis of Concern. Cache Valley virus is known to infect livestock, causing birth defects. There have been two reports of encephalitic disease in humans attributed to Cache Valley virus. This virus has been isolated from biologics manufacturing processes employing Chinese hamster cell substrates on a number of occasions from 2000 to 2004 (Nims et al., BioPharm. Int. 21: 89-94, 2008). The route of entry of the virus into biologics production processes has not been established with certainty, although the use of contaminated bovine serum is considered to be the most likely source. Thus far the virus has only been known to infect manufacturing processes employing bovine serum.
Regulatory Expectations. Cache Valley virus is not mentioned specifically in any regulatory guidance, as the detection of this virus in biologics production has been reported only within the past decade. It is the intent of the guidance, however, that occurrences of viral contamination in biologics manufacturing be dealt with through implementation of specific testing methods as required to assure detection of future recurrences (e.g., ICH Q5A R1). In addition, it is expected that the route of entry of the virus be established and that the process be remediated so that future recurrences are prevented where possible (e.g., 1997 Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use).
Mitigating Risk. Although many Contract Testing laboratories offer rapid nucleic acid-based detection assays for Cache Valley virus, raw material screening for this virus using such assays does not appear to be a viable means of eliminating risk. The industry experience thus far indicates that Cache Valley virus may be a low level, non-homogeneous contaminant of bovine serum. Viral screening performed on one bottle out of a large lot of serum therefore is no guarantee that this virus will not be encountered. Elimination of animal-derived materials (esp. bovine serum) from the manufacturing process may help to reduce the risk of experiencing this virus. Should this not be possible, treatment of the serum or serum-containing media should be considered. Gamma-irradiation has been demonstrated to be effective in inactivating this virus in bovine serum (Gauvin, 2009). UVC treatment of media containing bovine serum also appears to be quite effective at inactivating Cache Valley virus (Weaver, 2009).
Conclusions. Cache Valley virus infects livestock and has been found to contaminate biologics manufacturing processes employing bovine serum. It is a virus of concern for biologics manufacturers employing bovine serum which has not been gamma-irradiated. Risk of infection of biological products with Cache Valley virus through use of bovine serum may be mitigated through implementation of gamma-irradiation of the serum, or UVC- or high-temperature short-time (HTST) treatment of media containing the serum and of viral purification processes capable of removing and inactivating enveloped viruses.
Cache Valley virus is a single-stranded RNA virus of family bunyavirus, genus Bunyavirus. It is enveloped and nominally 80-120 nm in diameter. Cache Valley virus was first isolated in Utah in 1956 and is carried by mosquitoes. It has since been found to be a widespread virus, having been isolated in Texas, Michigan, North Carolina, Indiana, Virginia and Maryland, for example.
source: Nims et al., BioPharm. Int. 21: 89-94, 2008
Basis of Concern. Cache Valley virus is known to infect livestock, causing birth defects. There have been two reports of encephalitic disease in humans attributed to Cache Valley virus. This virus has been isolated from biologics manufacturing processes employing Chinese hamster cell substrates on a number of occasions from 2000 to 2004 (Nims et al., BioPharm. Int. 21: 89-94, 2008). The route of entry of the virus into biologics production processes has not been established with certainty, although the use of contaminated bovine serum is considered to be the most likely source. Thus far the virus has only been known to infect manufacturing processes employing bovine serum.
Regulatory Expectations. Cache Valley virus is not mentioned specifically in any regulatory guidance, as the detection of this virus in biologics production has been reported only within the past decade. It is the intent of the guidance, however, that occurrences of viral contamination in biologics manufacturing be dealt with through implementation of specific testing methods as required to assure detection of future recurrences (e.g., ICH Q5A R1). In addition, it is expected that the route of entry of the virus be established and that the process be remediated so that future recurrences are prevented where possible (e.g., 1997 Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use).
Mitigating Risk. Although many Contract Testing laboratories offer rapid nucleic acid-based detection assays for Cache Valley virus, raw material screening for this virus using such assays does not appear to be a viable means of eliminating risk. The industry experience thus far indicates that Cache Valley virus may be a low level, non-homogeneous contaminant of bovine serum. Viral screening performed on one bottle out of a large lot of serum therefore is no guarantee that this virus will not be encountered. Elimination of animal-derived materials (esp. bovine serum) from the manufacturing process may help to reduce the risk of experiencing this virus. Should this not be possible, treatment of the serum or serum-containing media should be considered. Gamma-irradiation has been demonstrated to be effective in inactivating this virus in bovine serum (Gauvin, 2009). UVC treatment of media containing bovine serum also appears to be quite effective at inactivating Cache Valley virus (Weaver, 2009).
Conclusions. Cache Valley virus infects livestock and has been found to contaminate biologics manufacturing processes employing bovine serum. It is a virus of concern for biologics manufacturers employing bovine serum which has not been gamma-irradiated. Risk of infection of biological products with Cache Valley virus through use of bovine serum may be mitigated through implementation of gamma-irradiation of the serum, or UVC- or high-temperature short-time (HTST) treatment of media containing the serum and of viral purification processes capable of removing and inactivating enveloped viruses.
Wednesday, December 23, 2009
What cell line is this anyway?
By Dr. Ray Nims
For about as long as scientists have been using cell cultures in biomedical research, there have been cases of cell line misidentification. This has been especially true for continuous cell lines, with the increased probability over time of mislabeling or cross-contamination. The primary cross-contaminant historically has been HeLa, a human cervical carcinoma cell which, given the opportunity, could outgrow most other cells in culture. More recently, the use of feeder cells for the propagation of human stem cells, and the use of xenografting for the propagation of human tumor cells, has provided additional opportunities for cell line cross-contamination and misidentification.
In the past, confirmation of cell line species of origin has been the main approach for authenticating cell lines. This was done initially by karyotyping or by immunological techniques, but more recently it has been done through the technique of isoenzyme analysis. An example of an isoenzyme analysis is shown below for Peptidase B and Aspartate Aminotransferase. These agarose gels show a positive control, a negative control (this is the band that does not line up with the others), the test article and a standard extract. These gels confirmed the identity of the test article as mouse derived, as expected.
Isoenzyme analysis has the advantage that it is rapid, not very technically demanding, and may be used not only to confirm species of origin but also to detect the presence of a cross-contaminating cell if the latter is present in the culture at 10% or greater (Nims et al., Sensitivity of Isoenzyme Analysis for the Detection of Interspecies Cell Line Cross-Contamination. In Vitro Cell. Dev. Biol.-Animal 34:35-39, 1998). In fact, isoenzyme analysis is currently the primary method employed within the biopharmaceutical industry for cell line authentication in satisfaction of 1993 Points to Consider and ICH Q5D guidance.
Recent advances in molecular diagnostic techniques have made possible the authentication of human cell lines to the individual level. DNA fingerprinting technologies have matured to the point that some of them, especially single nucleotide polymorphism (SNP) typing and single tandem repeat (STR) profiling, are now considered to be viable options for standardizing human cell authentication (see ATCC SDO newsletter article, page 5. For both human and animal cells, DNA fingerprinting provides a means of determining authenticity to the individual level. However, the primary drawback is that the fingerprinting techniques as routinely performed will be less or not at all useful for detecting interspecies cocultivations or cross-contaminations. For this purpose, it may be necessary to retain isoenzyme analysis as part of the authentication armament even when the molecular technologies become the definitive authentication practices for human and animal cell lines.
For about as long as scientists have been using cell cultures in biomedical research, there have been cases of cell line misidentification. This has been especially true for continuous cell lines, with the increased probability over time of mislabeling or cross-contamination. The primary cross-contaminant historically has been HeLa, a human cervical carcinoma cell which, given the opportunity, could outgrow most other cells in culture. More recently, the use of feeder cells for the propagation of human stem cells, and the use of xenografting for the propagation of human tumor cells, has provided additional opportunities for cell line cross-contamination and misidentification.
In the past, confirmation of cell line species of origin has been the main approach for authenticating cell lines. This was done initially by karyotyping or by immunological techniques, but more recently it has been done through the technique of isoenzyme analysis. An example of an isoenzyme analysis is shown below for Peptidase B and Aspartate Aminotransferase. These agarose gels show a positive control, a negative control (this is the band that does not line up with the others), the test article and a standard extract. These gels confirmed the identity of the test article as mouse derived, as expected.
Isoenzyme analysis has the advantage that it is rapid, not very technically demanding, and may be used not only to confirm species of origin but also to detect the presence of a cross-contaminating cell if the latter is present in the culture at 10% or greater (Nims et al., Sensitivity of Isoenzyme Analysis for the Detection of Interspecies Cell Line Cross-Contamination. In Vitro Cell. Dev. Biol.-Animal 34:35-39, 1998). In fact, isoenzyme analysis is currently the primary method employed within the biopharmaceutical industry for cell line authentication in satisfaction of 1993 Points to Consider and ICH Q5D guidance.
Recent advances in molecular diagnostic techniques have made possible the authentication of human cell lines to the individual level. DNA fingerprinting technologies have matured to the point that some of them, especially single nucleotide polymorphism (SNP) typing and single tandem repeat (STR) profiling, are now considered to be viable options for standardizing human cell authentication (see ATCC SDO newsletter article, page 5. For both human and animal cells, DNA fingerprinting provides a means of determining authenticity to the individual level. However, the primary drawback is that the fingerprinting techniques as routinely performed will be less or not at all useful for detecting interspecies cocultivations or cross-contaminations. For this purpose, it may be necessary to retain isoenzyme analysis as part of the authentication armament even when the molecular technologies become the definitive authentication practices for human and animal cell lines.
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