Safety Considerations of PERC6

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A. QC Testing of PER.C6 Cells for Use in the Manufacture of Biologicals and Vaccines

The safety of vaccines and biologicals manufactured in continuous cell lines of animal or human origin is of paramount importance and must be ensured by the manufacturer through a program of quality control (QC) testing applied to the product before release for human administration. This QC testing is intended to (i) ensure the identity of the product, (ii) ensure the safety and sterility of the product by demonstrating the absence of adventitious microbial agents, and (iii) ensure the safety and sterility of the product by demonstrating the absence of adventitious viral agents. The program for QC testing applied to a biological product, formalized as a release protocol, is developed as a responsibility of a Department of BioAnalytical Development. The release protocol is developed through an evaluation and integration of (i) relevant compendial literature and precedents, (ii) the origin of the cell line used for production and its development as a master cell bank, (iii) the sourcing and quality control testing of raw materials of animal origin used in manufacture, and (iv) the method of good manufacturing practice (cGMP) manufacture of the bulk and intermediate and final product considering, among other things, the quality of environment in which bioprocessing is conducted, the method of manufacture, in particular the isolation of the culture system from operators, and the consistency of preparation.

The release protocol prescribes the QC testing to be applied not only to final product but, importantly, master cell banks, master virus seeds, and other bioprocess inputs, raw materials of animal origin, and intermediate bulk products developed during downstream processing, purification and formulation. The release protocol specifies testing methods and volumes to be tested relying upon bacterial broth and agar cultures, embryonated eggs, small animals, and in vitro cell culture in a variety of primary and continuous cell lines of mammalian or human origin. These methods are well known to be sensitive to the detection of a variety of bacterial and viral agents and applied in concert provide a comprehensive and sensitive analytical approach upon which to ensure product safety. More recently, with the development of exquisitely sensitive polymerase chain reaction (PCR) methods for the detection of agents which are refractory to animal or cell culture, these classical propagation methods are commonly supplemented with agent-specific testing, using PCR and polymerase-enhanced reverse transcriptase (PERT) assays. The general methods of testing to ensure product safety are presented in illustrated form in Fig. 8.

1. QC Testing for the Release of PER.C6 Master Cell Bank

The development of PER.C6 research master cell bank (rMCB) A068-016 to support manufacture of biologies has been previously described. The release protocol to ensure the (i) identity, (ii) sterility, and (iii) viral safety of the rMCB is presented in Table II. The QC testing was conducted by contract at Inveresk Research (Tranent, Scotland) and at MicroSafe (Leiden, The Netherlands).

Table II

Release Protocol for Crucell rMCB A068-016

Table II

Release Protocol for Crucell rMCB A068-016

Test

Method

Identity

Isoenzyme analysis

Sterility

Broth and agar for cultivation of

bacteria, fungi, mycoplasma

In vitro indicator cells for detection of

mycoplasma using Hoechst stain

Viral safety

In vivo eggs

Eggs (allantoic and yolk sac)

In vitro cell culture

MRC-5, HeLa, Vero, bovine cells

Agent-specific testing

HBV, HCV, EBV, HHV6, HIV-1, HIV-2,

using PCR

HTLV-1, HTLV-2, AAV, B19, SV40

Agent-specific testing

PERT, S+L~, XC testing

for retroviruses

Method

Criteria for Evaluation

Turbidity, Colony Formation Cytoplasmic Fluoresence

Viability Gross morphology Hemagluttination

In Vivo Testing in Eggs

Injection of Eggs by Amniotic, Allantoic or Yolk Sac Routes with Observation for 7-14 days

In Vivo Testing in Animals

Injection of Adult or Suckling mice. Guinea pigs or Rabbits by IM. IP. or SC Routes with Observation for 7-60 days

Viability Fitness Evidence of Disease

In Vitro Testing in Cell Culture

Inoculation of Primary or Continuous Cell Lines of Human, Primate or Animal Origin with Observ ation for 14-28 days

Evidence of Cytopathology H emadsorption H emagluttination

Testing for Specific Virus Agents

Use of Sequence Specific Primers for PCR Amplification or PERT, or TEM

Evidence of Gene Specific Product

Evidence of Enzymatic Activity of RT

Figure 8 Testing methods for the demonstration of product safety.

Figure 8 Testing methods for the demonstration of product safety.

Sterility

Inoculation of Broth and Agar Culture and Cell Cultures with Observation of 14-21 days

2. QC Testing for the Release of a PER.C6 Working Cell Bank

The release protocol of research working cell bank (rWCB) A068-043W, according to the panel of testing, is presented in Table III. The QC testing was conducted by contract at Inveresk Research and at MicroSafe. This testing included tests for (i) identity, (ii) sterility, and (iii) viral safety in cells of human and simian origin.

3. Development of a Master Cell Bank at the Merck Research Laboratories

Cryopreserved vials of the rWCB were obtained from Crucell by the Merck Research Laboratories and expanded under conditions of cGMP manufacture to create a master cell bank (MCB) for future manufacturing use. This MCB has been released for use in the propagation of recombinant adenovirus according to a release protocol presented in Table IV. The preponderance of this QC testing was conducted by Q-One BioTech (Glasgow, Scotland).

This release protocol for the rWCB provides persuasive demonstration of the (i) identity, (ii) sterility, and (iii) viral safety of the PER.C6 MCB. This release protocol specifies animal testing in small animals to supplement the egg safety testing applied to the rWCB, expands the variety of primary and continuous cell lines used for viral safety using in vitro cell culture, and greatly broadens the variety of agent-specific testing using PCR-based testing and biochemical testing for retroviruses. The human cell line 293 was included in the panel of tissue culture cell lines in an attempt to detect the presence of any defective adventitious virus that requires the presence of El in the host cell. The direct assay for reverse transcriptase, as well as the detection of RT in cocultivation supernatant fluids, was done with the highly sensitive PCR-based reverse transcriptase (PBRT) assay. The supplemental PCR tests were included

Table III

Release Protocol for Crucell rWCB A068-043W

Test Method

Identity Isozyme

Sterility Broth and agar for cultivation of bacteria, fungi, mycoplasma

In vitro cell culture testing for mycoplasma

Viral Safety

In vitro cell culture Vero, MRC-5, PER.C6 Agent-specific testing Adeno-associated virus using PCR

Table IV

Protocol for Release for the a PER.C6 Master Cell Bank

Table IV

Protocol for Release for the a PER.C6 Master Cell Bank

Test

Method

Identity

Isozyme analysis DNA Fingerprinting

PCR-Based Test for El

Sterility

Broth and agar for cultivation of bacteria, fungi,

mycoplasma

In vitro cell culture testing for mycoplasma

Viral safety

In vivo eggs

Eggs (allantoic and yolk sac)

In vivo animals

Guinea pig, adult and suckling mouse

In vitro cell culture

VERO, MRC-5, 293, Rabbit Kidney 13, Vero,

bovine turbinate, porcine kidney

Agent-specific testing

Transmission electron microscopy PERT for RT

Raji, RD, H9 cell-cocultivation for retroviruses

PCR for HBV, HCV CMV, EBV, HHV6,

HHV7, HHV8, HIV-1, HIV-2, HTLV-1 &

HTLV-2, SiFV, SFV, AAV, B19, bovine

polyoma, SV40

with due consideration for the human origin of the cell line and the use of bovine serum for the derivation of the cell line. The tumorigenic potential of the cell line was tested beyond the anticipated manufacturing cell-passage level.

Satisfactory results were obtained from all QC testing. The results of the testings are presented in Table V.

  1. Tumorigenicity
  2. Tumorigenicity Studies of PER.C6 Cells

Three tumorigenicity studies were carried out on the PER.C6 cell line. The results of these studies are summarized in Table VI. In the first study, nude (nu/nu) mice were injected subcutaneously with 107 PER.C6 cells. Positive control animals were injected subcutaneously with 107 KB cells. KB is a known tumor-producing cell line derived from an epidermoid carcinoma (American Type Culture Collection; CCL-121). The study was conducted over 28 days, at which point all animals were necropsied and examined grossly and histologically. All of the positive control animals had growing nodules, and 8 of 10 male mice and 7 of 10 female mice receiving PER.C6 cells had growing nodules, thus producing a positive test (Table VIA).

At the time of the first study, 21 or 28 days was the duration that was usually used. Subsequently the Center for Biologies Evaluation and

Table V

Summary of Testing of PER.C6 Research Master Cell Bank (Passage No. 29)

Test

Specification

Result

Sterility (EP)

Mycoplasma (broth, agar and DNA staining)

In vitro virology for adventitious viruses (28 days, with cytopathic effect and haemadsorption) on Vero, MRC-5, HeLa and PER.C6 cells (PTC) Specific viruses

Human immunodeficiency virus types 1 and 2

Human T-lymphotropic virus types 1 and 2

Human hepatitis B + C Human cytomegalovirus Human parvovirus B 19 Human herpes virus 6 Simian virus 40 Adeno-associated virus Epstein-Barr virus

Bovine viruses (BVD, IBR and PI3) In vivo virology in suckling mice (i.e. and i.p.), and embryonated eggs, allantoic and yolk sac injections

Isoenzyme test for human origin

In vivo virology (adult mice, guinea pigs and suckling mice) and transmission electron microscopy (TEM)

Reverse transcriptase assay S+L~ focus forming assay and XC

plaque assay Tumorigenicity in nude mice Restriction analysis

Sequencing

Negative Negative

Negative

Negative

Negative

Negative Negative Negative Negative Negative Negative Negative Negative Negative

Confirmed

Absence of adventitious microbial contamination

Negative

Negative

Report result

No evidence of mutation or rearrangements

Report sequence

Negative Negative

Negative

Negative

Negative

Negative Negative Negative Negative Negative Negative Negative Negative Negative

Confirmed Free from infectious adventitious microbial contamination

Negative Negative

Tumorigenic

No evidence of mutation or rearrangements Sequence reported

(continued)

Table V (continued)

Test

Specification

Result

DNA profiling rMCB (passage 29) and late passage cells (passage 98)

Karyotyping/chromosomal analysis

Fluorescent product enhanced reverse transcriptase (PERT) assay

S+L~ focus forming assay and XC plaque assay

Multicolor fluorescent in situ hybridization (M-FISH)

Copy no. determination (fiber FISH analysis)

Prions

Determination of prions Sequence analysis

Late passage banding pattern resembles rMCB Report chromosome numbers Negative

Negative

Report integration site

Report results

No evidence for infectious PrPSc

Late passage banding pattern resembles rMCB

Modal No. 86. Range 68-106

Negative Negative Chromosome 14 13.6 ±6.1 Confirmed

Research (CBER) of the Food and Drug Administration had suggested the observation period be extended to 84 days. This was to give more time for slow growing tumors to appear and for nontumorigenic nodules to regress or disappear. Therefore, the tumorigenicity study on the PER.C6 cells was repeated.

The second study was performed in nude (nu/nu) mice over an 84-day period. Thirty nude mice were injected subcutaneously with 107 PER.C6 cells in 0.2 mL of serum-free medium. As a positive control, 10 mice were injected subcutaneously with 106 HeLa cells in 0.2 mL of serum-free medium. As a negative control, 30 mice were injected with 0.2 mL of medium. The mice were palpated at the injection site every 3 to 7 days and any nodules found were measured in two dimensions. The PER.C6 cell test arm and the negative control arm had 10 mice necropsied 21, 42, and 84 days postinjections. The positive control arm was necropsied at 42 days postinjection. Gross and histological examinations were performed on all injection sites and nodules if they appeared. During the initial days after injection, palpable nodules were present at the subcutaneous injection sites in all animals inoculated with PER.C6 cells. Between postinjection days 5 and 14, the detectable masses disappeared from the injection sites. However, in several of these mice, the masses subsequently reappeared by around day 21 and continued to enlarge until the animals were necropsied. Of the mice injected with PER.C6 cells, 5 of 10 sacrificed on day 21, 5 of 10 sacrificed on day 42, and 1 of 10

Table VI Tumorigenicity of PER.C6 Cells

A. Day 28 tumorigenicity of PER.C6 and KB cells in

nude mice

Cell type

No. of cells

Male

Female

KB

1 x 107

10/10

10/10

PER.C6

1 x 107

9/10

7/10

B. 84-Day tumorigenicity study of PER.C6 and HeLa cells

Cell type

No. of cells

Day 21

Day 42

Day 84

HeLa

1 x 106

NA

10/10

NA

PER.C6

1 x 107

5/10

5/10

1/10

Medium control —

1/10"

0/10

0/10

C. Titration tumorigenicity study of PER.C6 cells in

nude mice

Cell type

No. of cells

Day 21

Day 42

Day 84

PER.C6

1 x 103

0/10

0/10

0/10

PER.C6

1 x 105

0/10

0/10

0/10

PER.C6

1 x 107

5/10

9/10

7/106

Medium

0/10

0/10

0/10

Note. Details of the experiment are presented in Section 5B.

" Benign lung adenoma.

h Seven animals sacrificed, with tumors on day 56 and leaving

Note. Details of the experiment are presented in Section 5B.

" Benign lung adenoma.

h Seven animals sacrificed, with tumors on day 56 and leaving

sacrificed on day 84 (actually sacrificed on day 49 due to tumor size) had gross or microscopic evidence of a tumor (Table VI B). Histologically, these recurrent nodules were composed of sheets of large pleomorphic cells with numerous, sometimes abnormal, mitotic figures. These masses compressed the surrounding tissues but were not invasive. No tumors were observed outside the injection sites. The interpretation of the test is that PER.C6 cells are positive for tumorigenicity.

In view of the positive tumorigenicity results obtained following injection of 107 PER.C6 cells, a titration study was performed in which nude mice were injected with PER.C6 cells at doses of 107, 105, or 103 cells per animal. Mice were necropsied 21, 42, or 84 days postinjection. No animals receiving 103 PER.C6 cells had palpable masses at the injection site from the first palpation day until necropsy. None of these animals had gross or microscopic evidence of nodules or tumor cell collections at any necropsy time point. Two of the 30 mice receiving 105 PER.C6 cells had palpable nodules on postinjection day 3.

These masses disappeared by day 7 and did not recur. Gross and histological examination of the injection sites were negative at all necropsy time points. In the mice that received 107 PER.C6 cells, 29 of 30 animals had palpable nodules on Day 3 — some of which disappeared or became smaller but most of these recurred and grew progressively until necropsy. At necropsy, 5 of 10 mice on day 21 had tumors, 9 of 10 mice sacrificed on day 42 had tumors, and 7 of 10 in the group scheduled for day 84 had tumors but were sacrificed on day 56 because of tumor size (Table VI C). The histological and gross features of the PER.C6 cell tumors were similar to those described for the previous study (above). No metastatic nodules were found. Thus, the tumorigenicity studies of PER.C6 cells were positive at 107 cells per animal and negative at 105 and 103 cells per animal. This would indicate that not all of the PER.C6 cells are tumorigenic and/or a critical mass of tumorigenic cells are necessary for tumor formation.

2. Tumorigenicity Studies of Residual DNA from PER.C6 Cells

In view of the positive tumorigenicity studies with 107 PER.C6 cells, the oncogenic potential of residual DNA from these cells was tested in both nude mice and newborn hamsters. For these studies, DNA was isolated from passage 61 PER.C6 cells using standard procedures. The DNA preparation was shown to be of high molecular weight (average size ~100 kb) and devoid of significant protein or RNA impurities. In the nude mouse study, 20 female nude (nu/nu) mice were injected subcutaneously with 225 p.g of PER.C6 DNA (in a volume of 0.25 mL). For negative controls, two groups of 20 female mice each were injected subcutaneously with 0.25 mL of vehicle. Approximately 5 months after injection, the mice were necropsied and examined histologically for tumor growth. None of the mice in this study exhibited gross or microscopic evidence of tumors at the injection site. One treated mouse had a lymphoma at a distant site. However, nude mice — particularly females — are known to have a high incidence of spontaneous lymphoma [75-78], and the occurrence of a single lymphoma in 20 treated mice is consistent with the spontaneous incidence. Although the lymphoma was almost certainly a spontaneous event, a polymerase chain reaction (PCR) study was performed on the lymphoma DNA to determine if there was any evidence for the presence of the adenovirus El region — the transforming agent of PER.C6 cells. The study was negative, with a sensitivity of approximately one copy of El per 750 tumor cells. Previously, El expression has been shown to be necessary to maintain the transformed state of 293 cells, which, like PER.C6 cells, were transformed by El [79]. The results of the PCR analysis support the conclusion that the lymphoma was a spontaneous event, not induced by PER.C6 DNA.

A second tumorigenicity study using DNA from PER.C6 cells was carried out in newborn hamsters. Between 18 and 36 h after birth, female and male hamsters (28 total) were injected subcutaneously with approximately 100 |xg of PER.C6 DNA (in a volume of 110 |xL). Two groups of control hamsters (50, mixed sex, per group) were injected with 100 |xL of vehicle. Several pups in each group were lost due to maternal cannibalism, reducing the group sizes to 20 (11 female, 9 male) in the PER.C6 DNA group, 40 (19 female, 21 male) in control group 1, and 45 (27 female, 18 male) in control group 2. After weaning, the hamsters were palpated on a weekly basis. The hamsters were necropsied approximately 5 months after injection and examined grossly and histologically for tumor growth. One female hamster in control group 2 died approximately 21 weeks after injection of a malignant ovarian teratoma. No evidence of tumors was found in the 20 hamsters that were injected with PER.C6 DNA.

3. Concerns about Using a Tumorigenic Cell Substrate

The basis for concern about using a tumorigenic cell substrate to produce a vaccine includes three theoretical possibilities. First, DNA from the cells carrying a putative activated oncogene or cancer-causing mutation could be integrated into the recipient's genome and produce a tumor. Second, a transforming protein in the cells could be transmitted and result in a tumor. Third, an adventitious tumor virus may be present and could be transmitted to the recipient and produce a tumor.

Concerning residual DNA from a tumorigenic cell substrate, there have now been several reports demonstrating that DNA extracted from tumorigenic cell lines or tumors growing in vivo — and even purified activated oncogenes— do not produce tumors when injected into animals at levels up to 1000 |xg of DNA [80-87], The negative results obtained with PER.C6 DNA in nude mice and newborn hamsters are consistent with these findings. In the case of the PER.C6 studies, the amount of DNA injected (~100 or 225 |xg) represents a >106-fold excess compared to the amount of residual DNA present in a dose of vaccine produced on this cell substrate. Others have calculated that 100 pg of residual DNA from tumorigenic cells would be equal to less than a billionth of a tumor-producing dose [80-87],

The second concern, transforming proteins or growth factors, has been considered by a WHO study group to be significant only if they are continually produced by cells or have continued administration [80, 81]. The study group did not consider the presence of contaminating known growth factors, in the concentrations that they would be found, to constitute a serious risk in biological products prepared from continuous cell lines.

The third category of concern, viruses or other adventitial agents, does present a potential risk. This risk is greatest when primary cells are used because of the frequent need for newly acquired cells that require repeats of the extensive testing for adventitial agents. Human diploid cell lines and continuous tumorigenic cell lines are thoroughly and routinely tested for a wide variety of known and unknown adventitial agents in a series of in vitro and in vivo assays, thus providing adequate assurance that adventitial agents will not be transmitted.

C. Prion-Related Issues

It is now generally accepted that an abnormal form of the cell surface glycoprotein PrP, or prion protein, is the main infectious agent in transmissible spongiform encephalopathies like scrapie, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease (CJD) ([88] and reviewed in [89]). The abnormal form of PrP, called PrPsc or PrP-res, is characterized by a remarkable resistance to denaturing agents and to degradation by Proteinase K (Prot K). Diagnostic tests take advantage of this unusual stability that allows a distinction between PrPc and PrPsc using antibodies that recognize both forms of PrP (e.g., [90]).

Human prion diseases occur in sporadic, acquired or inherited forms with different clinical and pathological phenotypes (reviewed in [91]). In 1996 a new variant of CJD (vCJD) was reported in the United Kingdom in relatively young patients with clinical features different from the known CJD forms [92], It was also found by strain typing that the prion protein of these patients was indistinguishable from the one that causes BSE, thus raising the question whether vCJD could be acquired by consumption of meat from cattle suffering from BSE [93, 94]. The possibility of transmission of PrPsc from bovine to human raises safety issues for cultured cell lines used for the production of human drugs.

Therefore, PER.C6 cells were carefully examined for the PrP phenotype (see below) as well as genotype. It has been found that specific mutations in the PrP gene are associated with hereditary forms of human prion disease (reviewed in [89] and [91]). Furthermore, a common methionine/valine polymorphism at codon 129 of the PrP gene appears to be associated with phenotypic variability and susceptibility to sporadic and iatrogenic CJD. The vast majority of patients suffering from sCJD and also from vCJD were found to be homozygous for 129 M, whereas patients heterozygous at codon 129 were strikingly underrepresented [95—97], To examine whether the PER.C6 PrP gene contains any of the known mutations associated with susceptibility to prion diseases, the PER.C6 PrP gene was sequenced. For these sequencing studies, genomic DNA from PER.C6 cells was isolated, and used to amplify the PrP gene sequences by PCR. The resulting PCR product was cloned into a vector, and the PrP gene in each of 13 PrP-containing clones was sequenced by BaseClear (Leiden, The Netherlands). Five of these clones contained sequences coding for the 129 Methionine PrPc protein, while the other eight contained the 129 Valine PrPc sequence, demonstrating the heterozygosity at this position. To confirm this observation, the resulting PCR product was also sequenced. As expected, a double peak (g/a) was observed in the 129 codon at a position defining it as a valine (if the nucleotide is a guanine) or as methionine (if the nucleotide is an adenine). The PER.C6 PrP gene sequence was then compared to the wild-type sequence published in GenBank (Accession No. M12899) and was found to be identical to the wild type gene; thus, ruling out the possibility that these cells possessed a hereditary mutation that would be predisposing for prion diseases. The sequence also revealed that PER.C6 cells are heterozygous for methionine/valine at codon 129.

PrPc is constitutively expressed in adult brain [90, 98, 99] and at lower levels in other tissues like liver and spleen [100]. PrP expression has also been found in a variety of rodent and human cell lines. Our studies on PER.C6 and 293 cells have shown that these cells also express the cellular form of PrP. A validated Western blot analysis of Prot K-treated protein extracts of PER.C6 cells and their parental HER cells has failed to detect any Prot K-resistant forms of PrP at passages 33 and 36 of PER.C6 cells and passage 6 of their parental HER cells.

In addition to the sequencing of the prion gene and testing for the presence of abnormal prion protein in the PER.C6 cells at an early and late passage level of the culture, serum and trypsin batches that were used were traced to see if any were derived in the United Kingdom.

Finally, it has been possible to adopt the PER.C6 cells to serum-free suspension so that bovine sera can be completely avoided in the future if desired.

The above-mentioned characteristics of PER.C6 make it a safe manufacturing cell line in this respect.

  1. Genetic Characterization of PER.C6 Cells
  2. Sequence Analysis of El

The integrity of the E1A and E1B coding regions present in PER.C6 was tested by sequence analysis. This was done by bidirectional sequencing of PCR fragments generated from these regions, and the sequence of these fragments was compared to the original pIG.ElA.ElB sequence, the construct that was initially used in transfection.

No mutations, deletions, or insertions were detected between the sequence of the PCR fragments and pIG.ElA.ElB, indicating that no genetic alterations were introduced in the El A and E1B regions during transfection and subsequent culture of the cells.

2. Site of Integration of El

The chromosomal integration site of the plasmid pIG.ElA.ElB in PER.C6 was determined by using the multicolor fluorescent in situ hybridization (MFISH) technique in combination with the principle of combined binary ratio labeling (COBRA) [101]. This technique combines 24-color COBRA-MFISH

using chromosome-specific painting probes for all human chromosomes with plasmid probe (pIG.ElA.ElB) visualization (25th color).

The pIG.ElA.ElB integration site was determined using PER.C6 cells that are derived from the research master cell bank (passage number 29). Cells were analyzed at passage numbers 31,41, 55, and 99. Two hundred and fifty metaphases and interphases were studied.

  1. ElA.ElB integration was detected only on chromosome 14 (Fig. 9, see color insert) and in both sister chromatids of the chromosome in all PER.C6 passage numbers screened. Of the 47 metaphases and 203 interphases, 75-80% consisted of integration of pIG.ElA.ElB in one chromosome 14, whereas 20-25% consisted of integration in two chromosomes 14 [102].
  2. Copy Number of the El Construct

The number of copies of pIG.ElA.ElB present in the PER.C6 chromosome was studied by Southern blot analysis, dot bot analysis and fiber FISH analysis [102]. Southern hybridization revealed the presence of several integrated copies of pIG.ElA.ElB in the genome of PER.C6 [46].

In addition, dot blot analysis showed a pIG.ElA.ElB plasmid copy number of 19 ± 3 (research master cell bank) and 24 ± 16 (extended cell bank, passage number 99) per genome.

From the results it was concluded that PER.C6 consists of five to six copies of pIG.ElA.ElB per haploid genome.

Fiber FISH enables physical length measurements of in sfiw-hybridized DNA probes on linearized DNA fibers with a resolution equal to the theoretical length of a linearized DNA molecule according the model of Watson and Crick (1 kb is 0.34 |xm). Therefore, fiber FISH was conducted to measure the length of the integrated construct in the PER.C6 cell line at passage numbers (pns) 31, 41, and 99. Twenty fibers were measured. It was determined that pIG.ElA.ElB was integrated in tandem copies in chromosome 14 of PER.C6. The copy number of these in-tandem integrations was determined to be as follows: pn31, 13.6 ±6.1; pn41, 18 ±4.5; and pn99, 20.1 ±7.9.

  1. Chromosome Analysis
  2. C6 cells from cellular passages 44 and 66 were harvested for chromosome analysis to determine the modal chromosome number and the karyotype in a sample of metaphase plates. Cells were harvested, and slides were prepared and stained using a standard giemsa banding (GTG) technique. At each passage level, the chromosomes in 50 metaphase plates were counted. Also, full karyotypes were prepared from each passage level.

At passage level 44, the chromosome number ranged from 43 to 160. The mean number of chromosomes was 72 and the modal number was

61. All metaphase plates examined had structural chromosomal changes and rearrangements. A marker chromosome 19 with additional material in the long arm (19q+) was the most common alteration and was found in 14 of the 20 metaphase plates that were karyotyped.

At passage 66, the chromosome number ranged from 42 to 112. The mean number of chromosomes was 63 and the modal number was 64. All metaphase plates karyotyped again were found to have structural changes. The 19q+ was again the most common change, observed in 15 of 20 karyotypes. There was also a marker chromosome 11 with extra material in the short arm (11 p+) in 14 of the 20 karyotypes and a marker chromosome 9 with additional material in the short arm (9p+) in 8 of the 20 karyotypes.

Several of the markers differed at the two passage levels, but the most common marker, 19q+, was the same. The continuing changes seen as passage level increases is typical of heteroploid continuous cell lines.

  1. DNA Fingerprinting
  2. C6 cells were also analyzed on two occasions by DNA fingerprinting. DNA profile analysis of PER.C6 indicated no changes in the banding pattern obtained between the research master cell bank (pn 29) and an extended cell bank that was laid down at passage number 99. On a second occasion, a consistent DNA fingerprint was obtained between pn 45 and pn 67. There was no evidence of cross contamination with other cell lines.

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