PLEASE TELL ME WHICH INGREDIENT PROMPTED YOU TO ALLOW THEM TO PUT THIS IN YOUR BODY? WAS IT THE ABORTED KIDNEY CELLS OR MAYBE IT WAS THE ABORTED RETINAL CELLS? HMMM OR MAYBE IT WAS THE CITRIC ACID YOU WANTED INSTEAD OF EATIUNG AN ORANGE? OH POSSIBLY THE ETHANOL? WHATEVER PROMPTED YOU, I AM VERY INTERESTED IN YOUR ANSWER, PLEASE EXPLAIN IGNORANCE OR MAYBE STUPIDITY TO ME THAT PUT SUCH A FEAR OF DYING THAT YOU WOULD BE A LAB RAT WITH THINGS IN IT THAT CAN NOT ONLY BE DEADLY ON THEIR OWN, BUT BABY’S LIFE WAS TAKEN IN ORDER TO SAVE YOURS? AND YOU ARE OK WITH THAT? HMMM…
DO NOT ARGUE WITH ME OR TELL ME THAT I AM FABERCATING THESE TRUTH READ THE INGREDIENTS WHICH BY THE WAY CAME FROM THE FDA GOVERNMENT SITE AND EVERY DESCRIPTION CAME FROM SCIENTIST. SO PLEASE TELL ME AGAIN HOW GOD IS OK WITH KILLING INNOCENT CHILDREN AND HAVE THEIR CELLS THEIR DNA PUT IN YOU? PLEASE TELL ME. IT DOES NOT MATTER IF IT WAS FROM A FETUS MURDERED IN 1975 OR 1920 THAT WAS A BABY AND GODS CREATION!
YOU THINK I AM BEING HARD? YOU THINK I AM BEING UNFAIR? YOU THINK I AM BEING UNCHRISTLIKE? REMEMBER HE OVERTURNED TABLES AFTER MAKING A WHIP TOGETHER TO CHASE PEOPLE OUT OF THE TEMPLE OF GOD TWICE, AND YOU ARE CARRYING THE CELLS OF A BABY THAT WAS PUT TO DEATH BECAUSE YOU FEARED DYING FROM SOMETHING THAT HAS A .1% DEATH RATE? PEOPLE DIE IN CAR ACCIDENTS MORE THAN THIS!
WHAT ARE THE INGREDIENTS IN THE JANSSEN COVID-19 VACCINE? The Janssen COVID-19 Vaccine includes the following ingredients: recombinant, replication-incompetent adenovirus type 26 expressing the SARS-CoV-2 spike protein, citric acid monohydrate, trisodium citrate dihydrate, ethanol, 2-hydroxypropyl-β-cyclodextrin (HBCD), polysorbate-80, sodium chloride.
Recombinant DNA, molecules of DNA from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. Since the focus of all genetics is the gene, the fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate genes. Although it is relatively easy to isolate a sample of DNA from a collection of cells, finding a specific gene within this DNA sample can be compared to finding a needle in a haystack. Consider the fact that each human cell contains approximately 2 metres (6 feet) of DNA. Therefore, a small tissue sample will contain many kilometres of DNA. However, recombinant DNA technology has made it possible to isolate one gene or any other segment of DNA, enabling researchers to determine its nucleotide sequence, study its transcripts, mutate it in highly specific ways, and reinsert the modified sequence into a living organism.
What is Recombinant DNA?
Recombinant DNA, which is often shortened to rDNA, is an artificially made DNA strand that is formed by the combination of two or more gene sequences. This new combination may or may not occur naturally, but is engineered specifically for a purpose to be used in one of the many applications of recombinant DNA.
This article will go into further detail about what DNA is, how rDNA is made and what it can be used for.
Background Information on DNA
DNA, also known scientifically as deoxyribonucleic acid, has a double helix structure and contains a combination of the nitrogen bases: adenine, thymine, guanine and cytosine. Although these four bases are the same in all organisms, they can be paired together and arranged in an infinite number of ways, such that each organism has a unique combination for their DNA strands.
Recombinant DNA, or rDNA, is the term used to describe the combination of two DNA strands that are constructed artificially. Genetic scientists can do this to create unique DNA strand for different purposes, using several types of techniques.
Like naturally occurring DNA, recombinant DNA has the ability to produce recombinant proteins. It is often these proteins that play the key role in the application of recombinant DNA.
Formation of rDNA
In most cases, rDNA is created in a laboratory setting using a process of molecular cloning. This method allows in vivo DNA replication, in the living cells of the subject.
A cloning vector is a DNA molecule that replicates inside a living cell and is used to form rDNA. The cloning vector is usually a small part of a DNA strand that holds the genetic information that is needed for the replication of cells. Polymerase chain reaction (PCR) is another method that can be used to replicate a specific DNA sequence and create rDNA, which is used to replicate DNA in a laboratory test tube.
The standard method of making recombinant DNA involves:
- Choosing the appropriate host organism and cloning vector.
- Preparation of vector DNA and DNA to be cloned.
- Creation of recombinant DNA.
- Introduction of rDNA to host organism.
- Screening for rDNA with specific properties sought from host organisms.
Peter Lobban and A. Dale Kaiser were the first scientists to propose a technique to form recombinant DNA. This soon caught on to other scientists and in 1972 the first paper that detailed a new way to insert genetic information using E. coli was released. Soon after in 1973, several other papers followed building on the concept and adding techniques of construction and formation.
In 1978, Werner Arber, Daniel Nathans and Hamilton Smith were awarded the Nobel Prize in Medicine for creating technology to discover, isolate and apply rDNA. Since this time, recombinant genes and proteins have become widely used in medicine and agriculture. This offers a novel method of managing some health conditions, such as the use of recombinant insulin in diabetes, as well pest-control for gardens and farms.
- All Recombinant DNA Content
- Recombinant DNA Applications
- replication-incompetent adenovirus type 26 expressing the SARS-CoV-2 spike protein
Vaccines based on replication incompetent Ad26 viral vectors: Standardized template with key considerations for a risk/benefit assessment☆
Replication-incompetent adenoviral vectors have been under investigation as a platform to carry a variety of transgenes, and express them as a basis for vaccine development. A replication-incompetent adenoviral vector based on human adenovirus type 26 (Ad26) has been evaluated in several clinical trials.
The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety and features of recombinant viral vector vaccines. This paper reviews features of the Ad26 vectors, including tabulation of safety and risk assessment characteristics of Ad26-based vaccines.
In the Ad26 vector, deletion of E1 gene rendering the vector replication incompetent is combined with additional genetic engineering for vaccine manufacturability and transgene expression optimization. These vaccines can be manufactured in mammalian cell lines at scale providing an effective, flexible system for high-yield manufacturing. Ad26 vector vaccines have favorable thermostability profiles, compatible with vaccine supply chains.
Safety data are compiled in the Ad26 vaccine safety database version 4.0, with unblinded data from 23 ongoing and completed clinical studies for 3912 participants in five different Ad26-based vaccine programs. Overall, Ad26-based vaccines have been well tolerated, with no significant safety issues identified. Evaluation of Ad26-based vaccines is continuing, with >114,000 participants vaccinated as of 4th September 2020.
Extensive evaluation of immunogenicity in humans shows strong, durable humoral and cellular immune responses. Clinical trials have not revealed impact of pre-existing immunity to Ad26 on vaccine immunogenicity, even in the presence of Ad26 neutralizing antibody titers or Ad26-targeting T cell responses at baseline.
The first Ad26-based vaccine, against Ebola virus, received marketing authorization from EC on 1st July 2020, as part of the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen. New developments based on Ad26 vectors are underway, including a COVID-19 vaccine, which is currently in phase 3 of clinical evaluation.
The adenovirus type 26 (Ad26) wild type virus was first isolated in 1956 from an anal specimen of a 9-month-old male child . As described in that study, 4 different isolates were obtained from anal and throat swabs from different children, some of whom experienced mild self-limiting enteric infections. Although, none of the illnesses could etiologically be associated with the isolated adenoviruses, it suggests that wild type Ad26 can, presumably, cause asymptomatic or minor illness . Human Ad26 has been considered to be a low-prevalent adenovirus due to the low frequency of Ad26 neutralizing antibodies in various populations compared with human adenovirus type 5. For example, a seroprevalence study of the 51 human adenovirus serotypes known at the time showed that several serotypes from particularly subgroups B and D, including Ad26, were rare in a Belgian population , suggesting that vectors (rAd) derived from these serotypes might be useful alternatives to Ad5-based vectors for vaccine development, since for Ad5-based vectors it was shown that their high prevalence hampered their clinical use , , , , . More extensive seroprevalence and immunogenicity studies showed that while all of these vectors exhibited low seroprevalence, Ad26-based vaccine candidates were the most immunogenic in animals . Further studies have shown that, depending on geographical location, 10%–90% of people tested have neutralizing antibodies against Ad26. However, neutralization titers are low to intermediate compared with those observed for other adenovirus types , , , .
2.1. Ad26 vector development
Adenovirus genomes are linear, non-segmented double-stranded DNA molecules with inverted terminal repeat (ITR) sequences at each end. The vector system for replication-incompetent Ad26 vaccine vectors consists of an adaptor plasmid and a cosmid . The adaptor plasmid contains the left end of the genome containing the left ITR and the packaging signal. It also contains a transgene expression cassette in place of the E1 region and a ∼ 2.5 kb fragment downstream of the E1 region to enable homologous recombination with the cosmid. The cosmid contains the majority of the Ad genome, spanning from the pIX sequence to the right ITR, with a deletion of the E3 region and a modified E4 open reading frame 6 (E4orf6). Transfection into a suitable packaging cell line (HEK293 cells, PER.C6® cells) and subsequent homologous recombination of the adaptor plasmid and cosmid results in the generation of a replication-incompetent E1/E3-deleted Ad26 vector. Packaging cell lines like the HEK293 and PER.C6® cell lines contain the E1 region of adenovirus serotype 5 (Ad5). Because within the adenoviral life cycle, E1 protein 55 K and E4 protein Orf6 form a complex that is pivotal for high-level late-gene expression, the E4-Orf6 sequence of Ad26 is replaced by the corresponding sequence from Ad5 in the vector. This modification has previously been shown to be necessary to allow for the efficient production of rAd35 virus on Ad5 E1-complementing cells . Finally, compensation for the loss of E3 is not needed since the E3 proteins are not essential for adenoviral growth in vitro but are involved in down regulating cellular immunological response mechanisms in an attempt of the adenovirus to escape the host immune system .
Most adenovirus serotypes use the coxsackievirus-adenovirus receptor for attachment to the target cell , . In contrast, Ad26 has been reported to utilize CD46 as its primary cellular receptor , , but more recent reports indicated only a limited interaction between Ad26 and CD46, even showing evidence of a role for αvβ3 integrin for efficient transduction of epithelial cells, or interaction with sialic acid , . These data suggest that receptor usage by Ad26 might be host cell-type dependent in vitro , . Target cells in vivo in the natural host are not known, but Ad26 virus can infect a variety of cell types in vitro. Detailed studies dissecting the attachment, internalization and intracellular trafficking of adenoviral vectors have shown that Ad26, amongst others, accumulate in the late endosome to a larger extent and trigger innate immune pathways differentially compared with Ad5-based vectors . Whether and how these differences may translate into differential profiles of adaptive immunity against the vaccine antigen is not known.
2.2. Manufacturing, formulation and stability
Ad26 vector-based vaccines are manufactured using the E1-complementing PER.C6® cell line, a continuous, human cell line capable of supporting the manufacturing of replication incompetent adenoviral vectors . One of the key strengths of this cell platform is that the cells can grow in suspension in serum-free media to very high cell densities. Cell counts of 100 million cells/mL, with a high percentage of viable cells, can be reached within 10 days of cell culture. Janssen has taken advantage of the ability to grow the PER.C6® cell line at high cell densities in a so called “intensified process”. Cell-specific yields are in the same range as is generally achieved with other Adenoviral vectors and E1-complementing cell lines, therefore, due to the higher cell densities yields per volume unit are higher. The complete manufacturing process has now been scaled up to 1,000 L allowing manufacturing at a commercial scale.
While lyophilized vaccines are generally more heat-stable than non-lyophilized alternatives , liquid vaccine formulations may have several advantages over lyophilized vaccines, including ease of manufacture, packaging, and simple administration procedures . For Ad26-based vectors, progress in formulation development has allowed for long-term storage of product at 2–8 °C, enabling product distribution through existing vaccine supply chains. Assessments of robustness during storage, handling and distribution conditions have shown that recombinant Ad26 vectors can be maintained stable under frozen conditions or at 2–8 °C, and, furthermore, showed to be stable in-use with a syringe/needle, also when subjected to agitation or temperature excursions .
What is HEK293 cells, PER.C6® cells
293 and PER C6 Cell Lines Using AD5
HEK (human embryonic kidney) 293 – the number of aborted fetal experiments prior to establishing the cell line.
HEK 293 is used to deliver the lentivirus gene in Dr Yamanaka’s adult skin cell reprogramming, as a cell line and reagent for testing many research products and in the drug Xigris. But one would never know by looking at the package insert. In order to obtain the truth, Children of God for Life filed a Freedom of Information Act Request with the FDA.
A human cell line is used in the production of Xigris, as noted under FDA document, PC 3420 AMP, in the first paragraph, which states:
Xigris is a recombinant form of human activated protein C. An established human cell line possessing the complementary DNA for the inactive human protein C zymogen secretes the protein into the fermentation medium.
About the 293 Cell Line
The protein C is produced in the HEK 293 aborted fetal cell line. 293 cells are available from the American Type Culture Collection. There are variants of the cell line that derive from the parent.
ATCC Number: CRL-1573 Price: $167.00
Designation: 293 Depositors: F.L. Graham
Biosafety Level: 2 Shipped: frozen
Organism: Homo sapiens (human) Morphology: epithelial
Tissue: kidney; transformed with adenovirus 5 DNA
The 293 cell line is a permanent line of primary human embryonal kidney transformed by sheared human adenovirus type 5 (Ad 5)DNA. [RF32725]
The cells express the transforming gene of adenovirus 5. Although an earlier report suggested that the cells contained Adenovirus 5 DNA from both the right and left ends of the viral genome [RF32764], it is now clear that only left end sequences are present. [RF50113]
The cells express an unusual cell surface receptor for vitronectin composed of the integrin beta-1 subunit and the vitronectin receptor alpha-v subunit. [RF33793] Purified DNA from this line is available as ATCC 45504 (25 micrograms) and ATCC 45505 (100 micrograms).
The Ad5 insert was cloned and sequenced, and it was determined that a colinear seqment from nts 1 to 4344 is integrated into chromosome 19 (19q13.2). [RF50113]
United States Patent 5,681,932
Grinnell October 28, 1997
Inventors: Grinnell; Brian W. (Indianapolis, IN)
Assignee: Eli Lilly and Company (Indianapolis, IN)
Application No.: 458372 Filed: June 2, 1995
1. The recombinant human protein C molecule produced by inserting a vector comprising the DNA encoding human protein C into an adenovirus-transformed host cell then culturing said host cell under growth conditions suitable for production of said recombinant human protein C.
2. The recombinant human protein C molecule of claim 1 wherein the adenovirus-transformed host cell is selected from the group consisting of AV12 cells and human embryonic kidney 293 cells.
3. The recombinant human protein C molecule of claim 2 wherein the adenovirus-transformed host cell is an AV12 cell.
4. The recombinant human protein C molecule of claim 2 wherein the adenovirus transformed host cell is a human embryonic kidney 293 cell.
About the 293 Abortion
See FDA report, page 81, lines 14-22.
“So the Kidney material, the fetal kidney material was as follows. The kidney of the fetus was, with an unknown family history, was obtained in 1972 probably. The precise date is not known anymore.
The fetus, as far as I can remember was completely normal. Nothing was wrong. The reasons for the abortion were unknown to me. I probably knew it at the time, but it got lost, all this information.”
About the PER C6 Abortion and Cell Lines
See FDA transcript. Comments by Dr. Van Der Eb, Crucel, Nevada:
“So I isolated retina from a fetus, from a healthy fetus as far as could be seen, of 18 weeks old. There was nothing special with a family history or the pregnancy was completely normal up to the 18 weeks, and it turned out to be a socially indicated abortus – abortus provocatus, and that was simply because the woman wanted to get rid of the fetus.”
“The father was not known not to the hospital anymore, what was written down was unknown father, and that was, in fact, the reason why the abortion was requested.”
“There was permission, et cetera, and that was, however, was in 1985, ten years before this. This shows that the cells were isolated in October 1985, Laeiden University in my lab. At that time already ’85, I should say the cells were frozen, stored in liquid nitrogen, and in 1995 one of these was thawed for the generation of the PER.C6 cells.
“And this is the final slide just showing you some comparisons between 293 and PER.C6. Again, I remind you that both cell lines were made in my lab for different reasons. The objective, as I indicated, is for 293–was basic research, and we have done many different transformation studies after that, not transformation studies, but gene expressions studies with human embryonic kidney cells in the years following that up to now, I would say.
“PER C6 was made JUST FOR PHARMACEUTICAL MANUFACTURING OF ADENOVIRUS VECTORS As to RCA free, PER.C6 are RCA Free. The history documentation of the cell line has been carried out completely for PER. C6 and was not done at that time for 293.
“And then pharmaceutical industry standards. . . . I realize that this sounds a bit commercial, but PER C6 were made for that particular purpose.”
About the Cell Lines and Adenovirus (AD5) Safety issues
See FDA report.
Background: In 1954, during discussions surrounding the development of adenovirus vaccines for use in the military, the U.S. Armed Forces Epidemiology Board (AFEB) recommended the use of “normal cells” as the substrate for vaccine production rather than cell lines established from human tumors. This decision was based on concerns about the possibility that human tumor cells might be contaminated with occult oncogenic agents that might be transferred to vaccine recipients, an event which might in turn increase the risk of cancer and other neoplastic diseases in vaccinees. As evidenced by current regulatory guidelines and activities of control authorities worldwide, the precedent set in 1954 by the AFEB remains an important factor in the acceptance of all substrates for vaccine manufacture. Currently, the only cultured animal cells that have been used as substrates in U.S. licensed viral vaccines have been primary cells (e.g., derived from monkey, chick, mouse), diploid cell lines (e.g., WI38, MRC-5, FRhL-2), or immortalized (continuous), non-tumorigenic cell lines (e.g., VERO).
Over the past 47 years, two important factors have emerged that warrant serious consideration of the use of immortalized tumorigenic cell lines for viral vaccine production. The first of these factors is that certain novel virus vectors that are presently under development for high-priority target diseases, most notably AIDS, cannot feasibly be propagated in traditionally acceptable cell substrates. The second factor is that scientific understanding of neoplastic processes and viral-induced carcinogenesis has rapidly advanced, as has the ability to detect and identify infectious, oncogenic agents and other types of adventitious agents that may potentially contaminate cell substrates. These factors underscore the need for developing a regulatory framework in which the relative benefits and risks in using tumorigenic cell lines for vaccine production can be carefully and cautiously revisited.
FDA would like the VRBPAC to consider the potential risks in using two novel cell substrates, 293 cells and PER.C6 cells. These cell lines were developed by transforming human embryonic kidney cells (293) and human embryonic retinal cells (PER.C6) with the transforming early region 1 (E1) of adenovirus type 5 (Ad5). Since cell lines such as 293 and PER.C6 express the Ad5 E1 region, they are able to complement the growth of defective Ad5 vectors which have been “crippled” by deletion of E1. Defective Ad5 vectors have been engineered to express foreign genes such as those from human immunodeficiency virus (HIV), the causative agent of AIDS, and vectors of this type are thought to have significant potential for vaccine development because of their demonstrated ability to generate cell-mediated immune responses to HIV. However, a feature of regulatory importance associated with Ad5-transformed cells is their capacity to form tumors in immunodeficient animals such as nude mice.
In considering potential risks associated with the use of these so-called Designer Cell Substrates i.e., neoplastic cells derived from normal human cells transformed by defined viral or cellular oncogenes or by immortalizing cellular genes (e.g., telomerase) OVRR/CBER is considering the approach outlined below within the framework of a “defined-risks” assessment (see enclosed reference Lewis et al., “A defined-risks approach to the regulatory assessment of the use of neoplastic cells as substrates for viral vaccine manufacture”, In Evolving Scientific and Regulatory Perspectives on Cell Substrates for Vaccine Development. Brown, Lewis, Peden, Krause (eds.) Develop. Biol. Stand. [in press]). This framework is intended to examine, and wherever possible, to quantify the potential risk of “transmitting” the tumorigenic components of the cell substrate used for vaccine production, and determine whether that “transmission” might pose a risk, particularly an oncogenic risk, to vaccinees. Factors that could influence the risk associated with the use of Designer Cell Substrates include (1) the known mechanism of cell transformation leading to the development of tumorigenic cells; (2) residual cell substrate DNA; and (3) the presence of adventitious agents, especially oncogenic viruses. These three factors are discussed in more detail below.
Tumorigenicity of Adenovirus 5-Transformed Designer Cell Substrates
The purpose of tumorigenicity testing as applied to cell substrates used for viral vaccine manufacture is to discriminate between cells that have the capacity to form tumors and cells that do not form tumors. The potential risk of oncogenic activity is thought to be higher for cell substrates that have the capacity to form tumors, whereas the potential risk is thought to be low for cell substrates that are unable to form tumors. In considering the risk of tumorigenicity of Ad5-transformed Designer Cell Substrates, it is important to consider the molecular processes that determine the ability of the cells to form tumors.
Adenovirus 5 does not produce tumors when injected into rodents, but it does transform rodent cells in tissue culture. Like adenovirus 5 virions, adenovirus 5-transformed cells do not produce tumors when injected into immunocompetent adult rodents, but these cells can form tumors when injected into immunodeficient rodents such as nude mice. The tumor-forming capacity of Designer Cell Substrates that are produced by transforming normal human cells with adenovirus 5 can be evaluated by comparing them with adenovirus 5-transformed rodent cells. The adenovirus 5 early region 1 (E1) is composed of the transcription units E1A and E1B, which transform normal cells to neoplastic cells through a multi-step process. The E1A transcription unit immortalizes the cells and establishes those characteristics of the transformed cells that permit them to be eliminated by the antitumor defenses of immunocompetent rodents. During the transformation process, E1A sensitizes cells to apoptosis (programmed cell death) and increases their susceptibility to killing by natural killer cells, macrophages, and cytotoxic lymphocytes, as well as cytokines such as tumor necrosis factor (see Routes et al., 2000a, 2000b). The adenovirus E1B region alone is unable to immortalize cells, but its function during neoplastic transformation ensures cell survival by inhibiting virus-induced cell killing. Thus, the E1A region immortalizes cells and determines their limited capacity to form tumors in immunodeficient rodents, whose antitumor immune defenses are compromised. The complexity of these tumor-host processes and their action through nontransferable, immune mechanisms of the host implies that the capacity of adenovirus 5 E1-transformed mammalian cells to form tumors in immunodeficient rodents does not represent a risk factor for the manufacture of viral vaccines provided the cells can be shown to be devoid of adventitious agents (see additional discussion on adventitious agents below).
Several approaches can be considered in evaluating tumorigenicity of adenovirus 5-transformed human cell substrates. These approaches include demonstration that the tumor-forming capacity of the cells in rodents is adenovirus 5-like, and that the cells in the master cell bank are devoid of known and occult adventitious agents.
Potential Risks of DNA in Vaccines
Residual DNA in vaccines derived from tumorigenic cells, including those transformed by Ad5, can pose potential risks to the vaccine recipient in two respects: oncogenicity and infectivity. Each of these biological properties must be considered and evaluated for each cell substrate.
The oncogenic risk of cell substrate DNA has been considered to be due to several mechanisms. First, the residual DNA could have dominant activated oncogenes that could exert their effect following expression in recipient cells. In the case of Ad5-transformed cells, the dominant oncogenes would include the E1A and E1B genes. Second, the incoming DNA could integrate into the host genome in certain genes, such as the p53 gene or the retinoblastoma susceptibility (RB) gene, termed tumor suppressor genes, which are involved in cell cycle control among other cellular processes. Loss of function of tumor suppressor genes has been associated with certain human tumors. Third, integration of residual cell-substrate DNA could result in the activation of cellular regulatory genes by promoter/enhancer insertion, and this could result in the development of a neoplastic phenotype; this mechanism for tumor development was initially described in chickens for leukemia formation by avian leukosis viruses. Another result of integration that has been described is an increased methylation of adjacent DNA sequences as well as sequences on other chromosomes, although the consequences of such changes in methylation patterns to a cell are unknown.
The second biological activity of DNA that should be considered is its potential infectivity. If a genome of a DNA virus or the provirus of a retrovirus is present in the cell substrate used for vaccine manufacture, then the residual DNA has the potential, upon inoculation into the vaccine recipient, to produce infectious virus from this DNA and thus establish a productive infection.
The assessment of the risk of DNA both the oncogenic risk and the infectious risk needs to be considered both in terms of (1) the amount of residual DNA inoculated; and (2) the concentration of oncogene or infectious genome present in this DNA. One assumption is that the biological activity of any DNA administered is directly proportional to the amount of that DNA, and if the active component (oncogene or infectious genome) is carried as part of the cell-substrate DNA, the amounts of the oncogene or infectious genome will be present at a level of 10-5 to 10-6. This is because the haploid mammalian genome is 3 x 109 base pairs, whereas an average gene is between 3 x 103 and 104 base pairs. Thus, if the residual DNA is present at 10 ng, an oncogene in that DNA would be present at between 0.00001 and 0.0001 ng. Currently there are no data indicating that purified isolated oncogenes or any other DNA are biologically active at these levels.
It is also important to note that an additional safety margin for oncogenic activity is provided by the multi-step nature of cancer. This is because if more than one gene or event is required, the risk is diminished and is given by the product of the risk for each event. Thus, if the risk of a neoplastic event being induced by one oncogene is 1 in 106, then if two oncogenes are required, the risk is reduced to 1 in 1012.
Strategies that can be considered in evaluating residual DNA for vaccine products manufactured in adenovirus 5-transformed cell substrates include restricting the level of residual DNA to 10 ng or less. If these levels of residual DNA are not feasible, other methods can be considered to demonstrate the safety of higher levels of DNA, such as the inoculation of cell-substrate DNA into neonatal rodents. In the future, more sensitive animal models and in vitro assays may be developed to assess the oncogenic activity of DNA.
The experience in the early 1960s with SV40 contamination of poliovirus and adenovirus vaccines and the continuing questions regarding whether SV40 could be responsible for some human neoplasms underscore the importance of keeping viral vaccines free of adventitious agents. This is particularly important when there is a theoretical potential for contamination of a vaccine with viruses that might be associated with neoplasia.
It is unclear whether neoplastic cells have a greater or lower adventitious agent risk than other types of cells. Because they can be grown for long periods in tissue culture, there may be greater opportunities for any adventitious agents to be detected. Because neoplastic cells survive indefinitely, it is easier to qualify and bank cells that have passed all tests, especially as compared with primary cells (which are derived repeatedly from live tissue and must be re-qualified with each use). Moreover, many neoplastic cells can be grown in serum-free medium, potentially reducing the likelihood of contamination with bovine adventitious agents. However, if their growth in tissue culture is not well controlled, there may exist additional opportunities for contamination of cells with a longer lifespan. In cases of neoplastic cells for which the transforming event is unknown, there is also a theoretical possibility that transformation occurred as a result of a previous viral infection. Because some mammalian tumors and some cells transformed by viruses contain infectious virus, cells transformed by an unknown mechanism have a theoretical risk of containing a transforming virus. Cells for which specific knowledge of the transforming event exists (and can be shown not to be a virus that persists in the cells) may be more easily characterized than cells for which there is no specific knowledge of the transforming event (which could theoretically have been due to an infection with a known or an unknown virus).
Extensive adventitious agent testing is required for all cells that are proposed for use in vaccine production. This includes testing in various tissue culture systems, inoculation of animals followed by observation or detection of pathogen-specific antibodies, observation by electron microscopy, and molecular tests as is appropriate based on the history and type of cell to be tested. Specific polymerase chain reaction assays are used to rule out the presence of many different viruses. PCR-based reverse transcriptase assays are used to rule out the presence of retroviruses. The most sensitive of these assays include amplification steps, either as a result of viral growth in culture or in an organism, or molecular amplification such as in PCR.
Although Ad5-transformed cells are thought to be transformed by a known mechanism, the consequences of overlooking an occult oncogenic agent are significant. As these are the first cells in their class to be considered for vaccine production, evaluating them for the presence of occult oncogenic agents could enhance confidence in their use. One relevant animal assay that could be used for such an evaluation is the inoculation of cell-free lysates into susceptible newborn rodents, followed by observation for 5-6 months. This assay would detect most known tumor viruses, as well as potentially detect unknown tumor viruses.
Several promising areas of research suggest that experimental assays to detect unknown adventitious agents could soon become more generally available. As such assays become available, they could be considered for use in qualifying novel cell substrates, including neoplastic cell substrates.
Risk of Transmissible Spongiform Encephalopathy (TSE)
In addition to assessing the possibility of contamination of cell substrates with infectious virus, it is important to consider other agents such as the agent of TSE. There are several mechanisms by which vaccine cell substrates, including neoplastic cells, could theoretically become infected with a TSE agent. First, viral vaccines are developed and manufactured in cell substrates that may be derived from humans, and all human cells represent a finite possibility of being derived from individuals with a propensity to develop sporadic or familial Creutzfeld-Jakob disease (CJD). Although the mechanism by which such individuals develop CJD is not understood, CJD has in the past been transmitted to humans by biological products derived from CJD patients, such as human growth hormone and dura mater grafts. Second, vaccine cell substrates are usually exposed to products derived from cattle during tissue culture. Bovine spongiform encephalopathy (BSE) has been transmitted to humans in Europe in the form of variant CJD, possibly due to ingestion of infected beef. Under certain circumstances, cells in tissue culture can support the replication of certain TSE agents, although it is not known whether human cells in tissue culture can sustain the replication of the BSE agent. Assays to detect TSE/BSE agent contamination exist, but they may not be sensitive enough to exclude low levels of contamination. Until more is known about the replication of TSE/BSE agents in tissue culture and until more sensitive assays to detect these agents become available, the concern over the possible contamination of viral vaccines with TSE/BSE agents will be at least a theoretical consideration in vaccine development.
The use of immortalized, neoplastic human cells as substrates to develop recombinant viral vectors as vaccines also raises theoretical concerns with regard to possible contamination with TSE/BSE agents. These concerns include: (1) the implications of the possible presence of a prion protein (PrP)-encoding gene (PRNP) that is abnormal in the individual from whom the cells were derived; (2) the possibility that the genomic instability attendant with neoplastic processes could produce pathogenic alterations in the normal PRNP gene; (3) the possible exposure to agents of BSE present in bovine serum used in the propagation of the cells; (4) the possibility that an increased level of expression of either a normal or abnormal PRNP gene or other unknown factors in neoplastic cells might, upon exposure, sustain the replication of abnormal PrP proteins or otherwise contribute to the development of TSE in humans; and (5) the possibility that differences in the levels of expression of PRNP genes among clonal/subclonal populations of neoplastic cells may make evaluation of these potential risks more difficult. Since the lifetime risk of sporadic TSE in the population is about one case per ten thousand people (see Brown, P. et al., , Potential epidemic of Creutzfeldt-Jakob disease from human growth hormone therapy. N Engl J Med. 1985, 313:728-731), there is a finite risk that random tissue samples used for the development of neoplastic cell lines could contain abnormalities that might be associated with TSE transmission.
Several strategies can be considered for evaluation of neoplastic human cells for possible contamination with TSE/BSE agents. These strategies include a determination of the origin of the cells with respect to the possible family history and medical history of the donor regarding TSE risk factors and the identification and documentation of possible exposure of the cell line to bovine-derived materials, such as fetal bovine serum from countries with BSE. Further, two validated methods that could be used to evaluate the potential risk from the TSE agent include sequencing of the PRNP gene from neoplastic cell substrates and evaluating all such cell substrates by Western blot for the presence of protease resistant PrP. Additional studies may become feasible in the near future and may include evaluation of PRNP expression levels, determination of the ability of a cell substrate to support replication of the BSE agent, and evaluation of for the presence of infectious TSE agents by animal inoculation. As new assays for the detection and evaluation of TSE agents become available, they should be introduced as appropriate for cell substrate screening.
OVRR/CBER plans to present these issues to the FDA TSE Advisory Committee for a comprehensive discussion in the near future. In the interim, for cell substrates for which the presence of TSE could be a risk, sponsors should evaluate this issue by a combination of strategies, as may be technically feasible. Nevertheless, OVRR/CBER would like this Committee to be aware of and consider those issues related to the possible presence or exposure of cell substrates used for the development of viral vaccines to agents associated with TSE/BSE.
Recent animal experiments have demonstrated the utility of Ad5 vaccine vectors as means of stimulating cell-mediated immunity against HIV-1. Based on the review of available data, OVRR/CBER believes that there is an extremely low probability that residual DNA from the adenovirus 5-transformed human cells could transfer traits that could induce neoplastic development in vaccinees. OVRR/CBER also believes that such cells may be considered for the development of HIV vaccines, provided that the phenotype of the cells can be documented to be of an adenovirus 5 E1 type, and that appropriate testing rules out the presence of adventitious agents within the limits of state-of-the-art technology.
Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins.
Keywords: Cell culture, clotting factor, glycosylation, HEK 293, immunogenicity, monoclonal antibody, post-translational modification, recombinant protein, therapeutic glycoprotein, vaccine
Protein therapeutics (including monoclonal antibodies [mAbs], peptides and recombinant proteins) represent the largest group of new products in development by the biopharmaceutical industry (Durocher & Butler, 2009; Ho & Chien, 2014).
These products are produced in a wide variety of platforms, including non-mammalian expression systems (bacterial, yeast, plant and insect) and mammalian expression systems (including human cell lines) (Ghaderi et al., 2012). Importantly, the most appropriate expression system depends on the particular protein to be expressed. Mammalian expression systems are generally the preferred platform for manufacturing biopharmaceuticals, as these cell lines are able to produce large, complex proteins with post-translational modifications (PTMs; most notably glycosylation) similar to those produced in humans (Durocher & Butler, 2009; Ghaderi et al., 2012; Swiech et al., 2012). Moreover, in the case of mammalian cell lines, and animal cell lines in general, most proteins can be secreted rather than requiring cell lysis to extract with subsequent protein refolding (as is the case with bacteria/prokaryotes). The most common mammalian (non-human) cell lines used for therapeutic protein production include Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK21) cells and murine myeloma cells (NS0 and Sp2/0) (Estes & Melville, 2014). However, these non-human mammalian cell lines also produce PTMs that are not expressed in humans, namely galactose-α1,3-galactose (α-gal) and N-glycolylneuraminic acid (NGNA). Because humans possess circulating antibodies against both of these N-glycans, non-human cell lines are usually screened during their production to identify clones with acceptable glycan profiles (Ghaderi et al., 2010).
Human cell lines have the ability to produce proteins most similar to those synthesized naturally in humans, which may be an advantage compared with other mammalian expression systems (Ghaderi et al., 2010). In particular, the structure, number and location of post-translational N-glycans can affect the biologic activity, protein stability, clearance rate and immunogenicity of biotherapeutic proteins (Arnold et al., 2007; Ghaderi et al., 2010; Swiech et al., 2012).
The first human cell line, HeLa, was established in 1951 from a cervical cancer (Scherer et al., 1953). Human diploid cells were developed in the 1960s for vaccine manufacturing; however, concerns for a latent oncogenic agent in these cell lines (despite a lack of suggestive phenotypic characteristics) delayed their acceptance. Currently, human diploid cells are used in the manufacture of many viral vaccines (Petricciani & Sheets, 2008). However, due to their rapid growth, high protein yield, and the investment in system optimization, animal cells remained the substrate of choice for the production of recombinant proteins and mAbs (Petricciani & Sheets, 2008).
Today, advances in technology have allowed for increased productivity with human cell lines, and there are now approved recombinant biotherapeutic products produced from the human embryonic kidney 293 (HEK293) and fibrosarcoma HT-1080 cell lines (Beck, 2009; Casademunt et al., 2012; Dumont et al., 2012; Glaesner et al., 2010; Peters et al., 2010; Wraith, 2008; Zimran et al., 2013). Additional biotherapeutic products produced in the PER.C6, HKB-11, CAP and HuH-7 human cell lines are currently being evaluated (Enjolras et al., 2012; Estes & Melville, 2014; Jones et al., 2003; Mei et al., 2006; Swiech et al., 2011, 2015). This article is a narrative review of the cell lines (with a focus on human cell lines) used for production of biotherapeutic proteins, both approved and in development.
Non-human expression systems used to manufacture biotherapeutic products
Many non-human expression systems have been utilized in the production of currently approved biotherapeutic proteins (Table 1).
Non-human expression systems used in the production of biotherapeutics approved in the United States and Europea,b.
|Expression system||Biotherapeutic product||FDA approval||EMA approval|
|Cervical cancer vaccine||Approved||Approved|
|Asparaginase Erwinia chrysanthemi||Approved||NA|
|Collagenase Clostridium histolyticum||Approved||NA|
|Mammalian (non-human) cell lines|
FDA, US Food and Drug Administration; EMA, European Medicines Agency; NA, not approved; CHO, Chinese hamster ovary; BHK, baby hamster kidney.
aData obtained from publically available resources (October 2014); all approved products may not be included.
bReferences: (ABSEAMED®, 2012; ACTEMRA®, 2013; ACTILYSE®, 2014; ACTIVASE®, 2012; ADCETRIS®, 2013; ADVATE®, 2014; ALDURAZYME®, 2008; ARANESP®, 2006; ARCALYST™, 2008; ARZERRA®, 2014; AVASTIN®, 2010; AVONEX®, 2007; BENEFIX®, 2012; BENLYSTA®, 2011; CAMPATH®, 2014; CATHFLO® ACTIVASE®, 2010; CEREZYME®, 2010; CERVARIX®, 2012; CIMZIA®, 2013; CIMZIA®, 2014; CYRAMZA®, 2014; ELELYSO™, 2014; ENBREL®, 2010; ENTYVIO®, 2014a,b; EPERZAN™, 2014; Epoetin alfa HEXAL®, 2012; EPORATIO®, 2009; ERBITUX®, 2009; ERWINAZE®, 2011; EYLEA®, 2012; EYLEA®, 2013; FABRAZYME®, 2006; FERTAVID®, 2009; FOLLISTIM®, 2011; GAZYVA™, 2014; GAZYVARO®, 2014; Ghaderi et al., 2012; GONAL-F®, 2010; GRANIX™, 2014; HELIXATE® NexGen, 2010; HERCEPTIN®, 2010; HUMIRA®, 2008; HYLENEX®, 2012; ILARIS®, 2014; JETREA®, 2012; JETREA®, 2013; KADCYLA®, 2013, 2014; KOGENATE® Bayer, 2010; KRYSTEXXA®, 2013; LUMIZYME®, 2010; LUVERIS®, 2005; MABTHERA®, 2008; MENVEO®, 2010; METALYSE®, 2006; MYALEPT™, 2014; MYOZYME®, 2011; NAGLAZYME®, 2005; NOVOSEVEN®, 2006; NOVOTHIRTEEN®, 2012; NPLATE®, 2009; NULOJIX®, 2011; OBIZUR™, 2014; Office of Device Evaluation and Center for Devices and Radiological Health, 2001; OPGENRA®, 2014; ORENCIA®, 2012; OVIDREL®, 2014; OVITRELLE®, 2006; PERJETA®, 2013a,b; PREVNAR®, 2009; PROCRIT®, 2000; PROLIA®, 2010; PULMOZYME®, 2010; RAXIBACUMAB™, 2014; REBIF®, 2008; REFACTO AF®, 2014; REMICADE®, 2009; REOPRO®, 2013; RITUXAN®, 2014; ROACTEMRA®, 2013; SAIZEN®, 1987; SEROSTIM®, 1987; SIMPONI®, 2009; SIMULECT®, 2008; Somatropin Biopartners, 2013; STELARA®, 2013; Swiech et al., 2012; SYLVANT®, 2015; SYLVANT™, 2014; SYNAGIS®, 2009; TANZEUM™, 2014; tbo-filgrastim, 2012; THYROGEN®, 2010; TNKASE®, 2011; TRETTEN®, 2014; TYSABRI®, 2011; US Food and Drug Administration, 2010, 2011, 2012, 2013, 2014; Vectibix®, 2014; Victoza®, 2009; VIMIZIM®, 2014a,b; VORAXAZE®, 2012; XEOMIN®, 2014; XIAFLEX®, 2014; XOLAIR®, 2010; XYNTHA®, 2011; YERVOY®, 2011; ZALTRAP®, 2013a,b; ZEVALIN®, 2009).
Bacterial expression systems (e.g. Escherichia coli) possess the advantages of being straightforward to culture, with rapid cell growth and high yields. In addition, protein expression can be initiated through promoter induction by addition of lactose or the lactose analogue isopropyl-β-d-thiogalactopyranoside (IPTG; IPTG induces the promoters lac, tac and trc). However, such systems are unable to produce complex, mammalian-like glycosylation due to the absence of the necessary enzymatic components and the intracellular compartmentalization required (Ghaderi et al., 2012; Graumann & Premstaller, 2006). In addition, mammalian proteins produced in these systems often aggregate, forming inclusion bodies, due to the low solubility of mammalian proteins in prokaryotic cells and absence of appropriate protein chaperone systems. Proteins produced in bacterial expression systems must often be extracted from inclusion bodies and refolded. Bacterial systems are therefore generally used for production of non-glycosylated proteins, including some mAbs, hormones, cytokines and enzymes (Ghaderi et al., 2012; Graumann & Premstaller, 2006).
Similar to bacterial expression systems, yeast expression systems (e.g. Saccharomyces cerevisiae and Pichia pastoris) achieve rapid cell growth and high-protein yields with straightforward production scalability and without the need for animal-derived growth factors (Gerngross, 2004). Yeast cell lines may also be used to produce proteins that cannot be obtained from E. coli due to the problems associated with folding and stereochemistry (Gerngross, 2004). The key challenge associated with yeast expression systems is their production of high mannose residues within their expressed PTMs (50–200 vs three molecules in human cells, as part of either N– or O-linked glycan structures), which may confer a short half-life and render proteins less efficacious and even immunogenic in humans (Dean, 1999; Gemmill & Trimble, 1999; Gerngross, 2004; Lam et al., 2007; Mochizuki et al., 2001). The development of yeasts that have been genetically modified to address the issue of high mannose content has been reported (Chiba et al., 1998; Gerngross, 2004; Ghaderi et al., 2012; Hamilton et al., 2003). The expression of a fully humanized sialylated glycoprotein in glycoengineered yeast constitutes a major advance in the use of yeast expression systems for biopharmaceutical manufacturing (Hamilton & Gerngross, 2007).
Plant and insect cell expression systems are able to produce proteins with complex glycosylation patterns; however, the glycan structures produced are significantly different from those produced in humans (Ghaderi et al., 2012). Plants lack many of the key glycosylated residues present in humans, most notably sialic acids. In addition, they produce α1,3-fructose and β1,2-xylose, which are absent in humans and may be immunogenic (Ghaderi et al., 2012). Notably, in 2012, taliglucerase alfa (ELELYSO®; Pfizer, New York, NY) was approved by the US Food and Drug Administration (FDA) for the treatment of type 1 Gaucher disease. This therapy is produced using genetically modified carrot plant root cells that produce the enzyme with a human compatible glycan profile (ELELYSO™, 2014).
Insect cells infected with the viral vector baculovirus (baculovirus-insect cell expression system) can also efficiently express recombinant proteins, and these systems are mostly used for the development of virus-like particles and, subsequently, vaccines (Kost et al., 2005; Liu et al., 2013). However, although they produce N-glycan precursors, these are trimmed, resulting in either high mannose or paucimannose residues that do not develop further into terminal galactose and/or sialic acid residues (Kost et al., 2005). This is evidenced by the lack of either galactosyltransferase or sialyltransferase activity. As in plants, insect systems may also express the fucosylated α1,3-linkage (Staudacher et al., 1999). However, in recent years, there have been developments in the use of transgenic insect cells, with humanized protein glycosylation mechanisms (Kost et al., 2005).
The majority of currently licensed biotherapeutic products are produced in non-human mammalian expression systems (Table 1), as these systems are able to produce PTMs that (outside of a human expression system) most closely resemble those in humans (Ghaderi et al., 2010). These expression systems are used to produce mAbs, hormones, cytokines, enzymes and clotting factors (Ghaderi et al., 2012).
The most frequently used mammalian system is the CHO cell line, which is used in the manufacture of >70% of currently approved recombinant proteins (Butler & Spearman, 2014). This cell line has demonstrated several major advantages. First, CHO cells are able to grow in suspension culture (which enables large-scale production; other cell lines, such as insect cells, also have this ability) and serum-free chemically defined media (enabling reproducibility across different batches of cultures with a better safety profile than in media that contain human- or animal-derived proteins) (Kim et al., 2012; Lai et al., 2013; Rossi et al., 2012). Historically, CHO cells allowed gene amplification, resulting in a higher recombinant protein yield (up to the gram per liter range for some proteins) and specific productivity, which was previously an issue in other mammalian cell lines (Carlage et al., 2012; Kim et al., 2012; Yang et al., 2014a,b). Other advances, such as the creation of stronger expression units and advanced hosts, better selection strategies (e.g. through technologic advances in screening for high-productivity clones) and targeting the transgene to transcriptional hotspots (site-specific integration of transgenes), also contribute to the high protein yields attained from these cells (Kim et al., 2012). In addition, this expression system is highly tolerant to changes in pH, oxygen level, pressure or temperature during manufacturing (Ghaderi et al., 2012; Lai et al., 2013). Furthermore, due to the long period of time that this cell line has been used, there is a degree of familiarity with the CHO platform within development and manufacturing organizations, regulatory agencies, and suppliers (e.g. cell culture media suppliers), which could potentially decrease overall timelines. This familiarity may also be beneficial when assessing contaminant profiles (e.g. host cell proteins), which may be better characterized for CHO cells compared with newer cell lines.
The first recombinant biotherapeutic protein produced in CHO cells was tissue plasminogen activator, approved in 1986 (Kim et al., 2012). Therefore, the safety profile of CHO cells has been established for more than 20 years (Butler & Spearman, 2014; Kim et al., 2012). CHO cells have been shown to have reduced susceptibility to certain viral infections compared with other mammalian cell lines (e.g. the BHK cell line), and routine screening systems for adventitious agents are effective in detecting cell line infections (Berting et al., 2010). This reduced susceptibility may be due to the fact that many viral entry genes are not expressed in CHO cells (Xu et al., 2011). Further, there is perceived species barrier protection with the use of hamster-derived cells, reducing the potential risk of transfer of contaminating adventitious agents to humans (Berting et al., 2010; Swiech et al., 2012). However, many viruses have the ability to cross the species barrier and may still pose a risk (Pauwels et al., 2007).
Perhaps the most important advantage of CHO cells is that they are able to produce proteins with complex bioactive PTMs that are similar to those produced in humans. However, CHO cells are unable to produce some types of human glycosylation (CHO cells lack α[2-6] sialyltransferase α[1-3/4] fucosyltransferases) and they produce glycans that are not expressed in humans, namely α-gal and NGNA (Bosques et al., 2010; Dietmair et al., 2012; Ghaderi et al., 2012). Circulating antibodies against both of these N-glycans are present in humans, which may lead to increased immunogenicity and altered pharmacokinetics of these products when used in humans (Ghaderi et al., 2010; Padler-Karavani et al., 2008). Additional screening in CHO cells is required in order to isolate clones lacking the α-gal and NGNA glycans. This screening may result in otherwise productive clones needing to be discarded (Ghaderi et al., 2010). However, the attachment of non-human glycans may not be a concern for therapeutic proteins that do not require glycosylation, which illustrates the importance of considering the specific product molecule when choosing an appropriate cell line for production of a protein.
Other mammalian cell lines used for the production of biotherapeutic proteins include BHK-21 cells, used in the production of some coagulation factors such as factor VIII (Wurm, 2004). When murine myeloma cell lines (NS0 and Sp2/0) have been used historically, they have generally been used in the production of mAbs, for example, palivizumab and ofatumumab (Barnes et al., 2000; Butler & Spearman, 2014; Ghaderi et al., 2012). These myeloma cells were derived from immunoglobulin-producing tumor cells that no longer produced their original immunoglobulins; these cells possess the appropriate machinery for producing and secreting these proteins (Barnes et al., 2000).
For proteins produced in all of these non-human cell lines, as well as those produced in human cell lines, potential safety concerns arise from the possibility of process-related contaminants and immunogenicity (World Health Organization, 2013). Process-related contaminants may include infectious agents (viral, bacterial, fungal, etc.) with the potential to result in host infection, nucleic acid contaminants with the potential to integrate into the host genome (theoretical), and other contaminants from the manufacturing process, such as exogenous non-human epitopes (e.g. from animal serum used during the manufacturing process) that can be incorporated into human cells and the resultant biotherapeutic protein (Ghaderi et al., 2012).
Human cell lines used to manufacture licensed products
HEK293 and HT-1080 are the two human cell lines most often used in the production of biotherapeutic proteins, which offer the advantage of producing fully human PTMs (Tables 2 and and3)3) (Loignon et al., 2008; Swiech et al., 2012).
Human cells lines and their therapeutic protein productsa,b.
|Cell line||Product||Indication||FDA approval status||EMA approval status|
|Drotrecogin alfa||Severe septicemia/septic shock||Approved 2001; withdrawn 2011||Approved 2002; withdrawn 2011|
|rFVIIIFc||Hemophilia A||Approved 2014||Submitted 2014|
|rFIXFc||Hemophilia B||Approved 2014||NA|
|Dulaglutide||Type 2 diabetes||Approved 2014||Submitted 2014|
|Human-cl rhFVIII||Hemophilia A||Submitted to the FDA||Approved 2014|
|Agalsidase alfa||Fabry disease||NA||Approved 2001|
|Epoetin delta||Anemia secondary to chronic renal failure||NA||Approved 2002; withdrawn 2009 (Europe)|
|Idursulfase||Hunter syndrome||Approved 2006||Approved 2007|
|Velaglucerase alfa||Type 1 Gaucher disease||Approved 2010||Approved 2010|
|CL184||Rabies virus infection||Submitted to the FDA||NA|
|MOR103||Rheumatoid arthritis, multiple sclerosis||Phase 1 clinical development||Phase 1 clinical development|
FDA, US Food and Drug Administration; EMA, European Medicines Agency; HEK, human embryonic kidney; NA, not approved; rFVIIIFc, recombinant factor VIII Fc fusion protein; rFIXFc, recombinant factor IX Fc fusion protein; rhFVIII, recombinant human factor VIII.
aData obtained from publically available resources (October 2014); all approved products may not be included.
bReferences: (ALPROLIX®, 2014; Bakker et al., 2005; Behrens et al., 2014; Casademunt et al., 2012; DYNEPO®, 2007; ELAPRASE®, 2012, 2013; ELOCTATE®, 2014; European Medicines Agency and Committee for Medicinal Products for Human Use, 2014; Glaesner et al., 2010; Octapharma, 2014; REPLAGAL®, 2006; TRULICITY™, 2014; VPRIV®, 2010a,b; XIGRIS®, 2008).
Comparison of human cell lines with other expression systems in the production of therapeutic proteins.
|• Absence of potentially immunogenic PTMs due to human-compatible glycosylation • Easily grown in suspension serum-free culture • Achieve rapid reproduction • Amenable to a number of transfection methods||• Clinical experience is not as extensive as for other cell lines, although experience is growing • Potential susceptibility to human viral contamination|
HEK293 cells are easily grown in suspension serum-free culture, reproduce rapidly, are amenable to a number of transfection methods, and are highly efficient at protein production (Swiech et al., 2012; Thomas & Smart, 2005).
HEK293-H (Berkner, 1993) and 293-F (Vink et al., 2014) cell lines are clonal isolates of the HEK293 cell line that were selected for fast growth in serum-free medium, superior transfection efficiency, and a high level of protein production (Gibco, 2014). Subclone 293-H also has improved adherence to monolayer culture (when serum-supplemented media are used) compared with other cell lines. Other modified HEK293 cells include the HEK293-T cell line and HEK293-EBNA1 cells. The HEK293-T (293-T) cell line expresses the simian virus 40 large T antigen and is capable of expressing high titers of viral gene vectors for use in gene therapy (Yamaguchi et al., 2003). HEK293-T cells are often used for the production of retroviral vectors (Yamaguchi et al., 2003). HEK293-EBNA1 cells stably express the Epstein-Barr virus EBNA-1 gene, controlled by the cytomegalovirus promoter and demonstrate a greater growth rate and maximal cell density relative to parental HEK293 cells (Schlaeger & Christensen, 1999).
HEK293 cells have been widely used to produce research-grade proteins for many years and, more recently, five therapeutic agents produced in HEK293 cells have been approved by the FDA or the European Medicines Agency (EMA) for therapeutic use. These agents are drotrecogin alfa (XIGRIS®; Eli Lilly Corporation, Indianapolis, IN), recombinant factor IX Fc fusion protein (rFIXFc; Biogen, Cambridge, MA), recombinant factor VIII Fc fusion protein (rFVIIIFc; Biogen, Cambridge, MA), human cell line recombinant factor VIII (human-cl rhFVIII; NUWIQ®; Octapharma, Lachen, Switzerland) and dulaglutide (TRULICITY®; Eli Lily, Indianapolis, IN).
Drotrecogin alfa is a recombinant activated protein C that was approved by the FDA in 2001 and by the EMA in 2002 for the treatment of patients with severe sepsis. HEK293 cells were chosen by the manufacturer for production of drotrecogin alfa because its activity required two PTMs, propeptide cleavage and γ-carboxylation of its glutamic acid residues, which CHO cells cannot produce with adequate efficiency (Berkner, 1993; Durocher & Butler, 2009). The product was approved (Bernard et al., 2001), but was later voluntarily withdrawn from the market by its manufacturer (Eli Lilly) in 2011 following the randomized placebo-controlled Prospective Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis and Septic Shock (PROWESS-SHOCK) trial, which demonstrated no mortality benefit with drotrecogin alfa compared with placebo for patients experiencing septic shock (Green et al., 2012; Ranieri et al., 2012).
rFVIIIFc and rFIXFc are recombinant fusion proteins that were approved by the FDA in 2014 for the control and prevention of bleeding episodes, perioperative management and routine prophylaxis to prevent or reduce the frequency of bleeding episodes in people with hemophilia A and B, respectively (ALPROLIX®, 2014; ELOCTATE®, 2014; Mahlangu et al., 2014; Powell et al., 2013). They are also approved in Canada, Australia and Japan. rFVIIIFc consists of B domain–deleted recombinant factor VIII genetically fused to the Fc portion of immunoglobulin G1 (IgG1) and is produced in HEK293-H cells (Dumont et al., 2012; ELOCTATE®, 2014; Peters et al., 2013). The rFVIIIFc fed-batch culture process is robust at scales of 200, 2000 and 15 000 liters, with the potential for a second-generation process to achieve even higher cell densities, on the order of 3.5 × 107 vc/ml (Huang et al., 2014). rFIXFc was also produced using HEK293-H cells, and consists of the factor IX sequence covalently linked to the Fc domain of human IgG1 (ALPROLIX®, 2014; Durocher & Butler, 2009; McCue et al., 2014; Peters et al., 2010). An essential PTM for FIX activity is γ-carboxylation of the first 12 glutamic acid residues in the Gla domain by vitamin K–dependent γ-glutamyl carboxylase. This modification facilitates binding of FIX to phospholipid membranes. HEK293 cells have been reported to have a greater capacity for γ-carboxylation than CHO cells (Berkner, 1993). Furthermore, FVIII contains six potential tyrosine sulfation sites, which are vital for FVIII functionality and binding to von Willebrand factor. FVIII expressed from human cell lines has been reported to be fully sulfated (Kannicht et al., 2013; Peters et al., 2013).
The use of a human cell line for replacement coagulation factors, such as rFVIIIFc and rFIXFc, may result in reduced immunogenicity relative to non-human mammalian cell lines, as α-gal and NGNA glycan moieties are absent from these manufactured protein products (Bosques et al., 2010; McCue et al., 2014, 2015; Noguchi et al., 1995). However, it should be noted that several recombinant clotting factor products produced in non-human mammalian cell lines have been used successfully for many years. The development of inhibitors (neutralizing antibodies) against replacement clotting factors occurs in ∼30% of people with severe hemophilia A and 5% of those with severe hemophilia B. The causative F8 or F9 gene mutation plays a pivotal role in inhibitor development in hemophilia A and B, respectively, with large or complete deletions, non-sense mutations or inversions (e.g. intron 22 inversion in the F8 gene) being the most commonly associated mutations (Franchini & Mannucci, 2011). The impact of PTMs on inhibitor development is unknown, and will need further research. Importantly, none of the previously treated people with hemophilia in the phase 1/2a or phase 3 clinical studies developed inhibitors to the rFVIIIFc and rFIXFc fusion products (Mahlangu et al., 2014; Powell et al., 2012, 2013; Shapiro et al., 2012).
Human-cl rhFVIII (NUWIQ®), an additional factor VIII replacement product for the management of hemophilia A, is being produced in the HEK293-F cell line. Like HEK293-H cells, HEK293-F cells are a derivation of HEK293 cells that have been pre-adapted for growth in serum-free culture medium (Casademunt et al., 2012). Human-cl rhFVIII has been approved by the EMA and submitted to the FDA for approval (Octapharma, 2014); this product has been shown to exhibit a similar glycosylation profile to human plasma-derived factor VIII, without α-gal and NGNA (Kannicht et al., 2013).
Glucagon-1-like peptide (GLP-1) Fc fusion protein (dulaglutide) has been approved by the FDA for the treatment of type 2 diabetes mellitus, and is produced using HEK293-EBNA cells (Glaesner et al., 2010; TRULICITY™, 2014). Large clinical trials have demonstrated its superiority over the dipeptidyl peptidase-4 inhibitor antagonist exenatide and its non-inferiority to liraglutide (a GLP-1 agonist), when added on to oral diabetic agents (Dungan et al., 2014; Wysham et al., 2014).
Another human cell line, HT-1080, was produced from a fibrosarcoma with an epithelial-like phenotype (Swiech et al., 2012). With the use of gene activation technology (in which the endogenous DNA promoter is replaced with a more potent type), four approved therapeutic proteins have been produced by Shire (Swiech et al., 2012).
1) Epoetin delta (DYNEPO®) was approved by the EMA in 2002 for the treatment of anemia secondary to chronic renal failure (DYNEPO®, 2007; ELAPRASE®, 2013; REPLAGAL®, 2006; Swiech et al., 2012; VPRIV®, 2013). However, this has been voluntarily withdrawn by the manufacturer for commercial reasons.
2) Iduronate-2-sulfatase (idursulfase; ELAPRASE®) is licensed as enzyme replacement therapy (EMA in 2007 and FDA in 2006) for the treatment of Hunter syndrome (mucopolysaccharidosis II), an X-linked lysosomal storage disorder (ELAPRASE®, 2013).
3) Agalsidase alfa (REPLAGAL®; Shire Human Genetic Therapies, Danderyd, Sweden) was approved by the EMA in 2001 for the treatment of Fabry disease (REPLAGAL®, 2010). Compared with agalsidase beta (FABRAZYME®; Genzyme Therapeutics, Cambridge, MA), which is produced using CHO cells for a similar indication (FABRAZYME®, 2010, 2014), agalsidase alfa has shown similar enzyme kinetics. However, agalsidase alfa demonstrates a lesser uptake into fibroblasts from patients with Fabry disease and also lower concentrations in the kidney, heart and spleen of mice (Lee et al., 2003). A single clinical study has compared the two products; this showed no significant differences for all efficacy outcomes, and there were no differences for the development of antibodies (Vedder et al., 2007).
4) The fourth agent produced in HT-1080 cells, velaglucerase alfa (VPRIV®; Shire Human Genetic Therapies, Lexington, MA), was approved in 2010 (FDA and EMA) for the treatment of type 1 Gaucher disease (DYNEPO®, 2007; ELAPRASE®, 2013; REPLAGAL®, 2006; Swiech et al., 2012; VPRIV®, 2013). Velaglucerase alfa has been compared with two similar products: imiglucerase, produced using CHO cells, and taliglucerase alfa, produced using carrot cells (Ben Turkia et al., 2013; Tekoah et al., 2013).
These products have diverse glycan profiles and the studies have generally shown comparable uptake into macrophages, in vitro enzymatic activity, stability, organ distribution and efficacy (Ben Turkia et al., 2013; Tekoah et al., 2013). However, neutralizing antibodies to imiglucerase were noted in 24% of patients, which had an impact on enzyme activity. It was noted that various factors, such as the production cell line and glycosylation, may be responsible for the difference in immunogenicity, however, the specificity of the anti-imiglucerase antibodies was not stated (Ben Turkia et al., 2013).
Notably, studies that evaluated epoetin delta produced in HT-1080 cells demonstrated differences in glycosylation compared with erythropoietin produced in CHO cells, including a lack of NGNA in the proteins (Butler & Spearman, 2014; Llop et al., 2008; Shahrokh et al., 2011). However, there were additional overlapping isoforms present in endogenous human erythropoietin isolated from urine and serum relative to epoetin delta that could not be accounted for by sialic residues alone.
Human cell lines used in the expression of proteins in clinical and preclinical development
Human cell lines have been extensively utilized for the production of products that are currently in clinical development. In addition, human cell lines are a frequently used expression system for biomedical research due to their production of human PTMs and high productivity. As productivity may vary across clonal isolates, it is important to screen for those clones with the highest yield of the therapeutic protein (Berkner, 1993).
The PER.C6 cell line was created from human embryonic retinal cells, immortalized via transfection with the adenovirus E1 gene (Havenga et al., 2008). This system was originally developed for the production of human adenovirus vectors for use in vaccine development and gene therapy (Butler & Spearman, 2014). An investment was made in this cell line in order to develop a human expression system, and now an advantage of PER.C6 is its ability to produce a high level of protein when used in the production of human IgG (Jones et al., 2003). However, this does not require amplification of the incorporated gene (Jones et al., 2003). Currently, a variety of products utilizing the PER.C6 cell line are in phase 1 or 2 clinical trials (Durocher & Butler, 2009), including the MOR103 mAb, a human IgG antibody against granulocyte macrophage colony-stimulating factor, and CL184, an antibody against the rabies virus (Nagarajan et al., 2014).
MOR103 is in clinical development for the treatment of rheumatoid arthritis and multiple sclerosis. In a phase 1b/2a, randomized, placebo-controlled study, MOR103 was active in patients with moderately severe rheumatoid arthritis; a small number of patients developed anti-MOR103 antibodies (Behrens et al., 2014). CL184 is a combination of two mAbs, human IgG1(λ) and human IgG1(κ) (Bakker et al., 2005). In a phase 1 clinical study, it demonstrated a favorable safety profile and rapid development of rabies virus neutralizing activity, while there was no evidence to suggest the development of human anti-human antibodies (Bakker et al., 2008). CL184 has been granted FDA fast-track approval status.
Two additional cell lines are utilized by products currently in preclinical development. The CAP cell line is derived from human amniocytes obtained through amniocentesis; these cells are immortalized through an adenovirus type 5 E1 gene (Schiedner et al., 2008; Swiech et al., 2011). In addition to the ability to produce human PTMs, the primary advantage of this cell is the potential for high protein yields (Schiedner et al., 2008).
The HKB-11 cell line was created through polyethylene glycol fusion of HEK293-S and a human B-cell line (modified Burkitt lymphoma cells) (Cho et al., 2003; Durocher & Butler, 2009; Picanco-Castro et al., 2013). The advantages of this cell line include high-level protein production without the formation of aggregates, which can be a problem in other human cell lines (Picanco-Castro et al., 2013). Notably, HKB-11 has demonstrated increased expression of human FVIII compared with expression in HEK293 and BHK21 (Mei et al., 2006). Similar to other human cell lines, it has been shown to produce human glycosylation patterns including α (2,3) and α (2,6) sialic acid linkages (Picanco-Castro et al., 2013). HKB-11 has been used to produce a recombinant factor VIII protein and tissue factor (Cho et al., 2003).
A more recently developed cell line, HuH-7, originates from a human hepatocellular carcinoma (Enjolras et al., 2012). A recent study has shown that the HuH-7-CD4 clone is capable of producing recombinant human factor IX with a human glycosylation profile. PTM profiles (e.g. glycosylation, sialylation, phosphorylation and sulfation) were similar to plasma-derived and recombinant factor IX (rFIX), and were improved relative to rFIX produced in CHO cells (Enjolras et al., 2012). More recently, the HuH-7 cell line has been used to produce mutant forms of rFIX that have improved binding affinity for activated FVIII, and also demonstrated enhanced clotting activity in mice (Perot et al., 2015).
Perceptions of risks versus benefits of using human cell lines
The human-specific glycosylation pattern of the PTMs produced by human cell lines offer several advantages compared with those produced in animal cell lines. Although other mammalian cells can produce similar PTMs to human cells, most also produce α-gal and NGNA, PTMs that are not present in the structure of human proteins (Ghaderi et al., 2012). Patterns of post-translational glycosylation are known to affect protein yield, bioactivity, and clearance (Ghaderi et al., 2010). In addition, antibodies to NGNA have been widely reported to occur in humans (Chung et al., 2008; Ghaderi et al., 2012). One study utilizing an NGNA knockout mouse model demonstrated increased immunogenicity of cetuximab due to anti-NGNA antibodies (Ghaderi et al., 2010). In addition, in patients receiving the mAb cetuximab for the treatment of colorectal or head and neck cancers, the majority of severe hypersensitivity reactions observed in clinical trials were associated with pre-existing IgE antibodies against α-gal (Chung et al., 2008; Ghaderi et al., 2012). Such antibodies may alter the efficacy or immunogenicity of proteins with the presence of non-human glycan structures. Thus, human cell lines can serve as a valuable niche expression system for biotherapeutic proteins that require human PTMs. A theoretical concern with the use of human cell lines is an increased risk of transfer of human adventitious agents, given the lack of a species barrier (Swiech et al., 2012). However, current manufacturing technologies, typically inclusive of multiple viral inactivation or clearance steps, such as nanofiltration, have largely mitigated this concern and may provide more effective viral clearance than has been observed in CHO cells (Kelley et al., 2010; McCue et al., 2014, 2015).
Production of biotherapeutic proteins in human cell lines is expanding, with several products currently approved for clinical use and others in clinical development in different therapeutic areas. Advantages of human expression systems include achieving equal productivity to other mammalian cell lines and the production of proteins that lack potentially immunogenic, non-human PTMs (most notably α-gal and NGNA). In the future, with additional research investments and a continuation of the technologic advances that have already led to improvements in the use of human cell lines for protein manufacture, human cell lines will be further optimized, more sophisticated product collection strategies will be developed, and these cell lines may become one of the preferred platforms for protein biotherapeutic production.
What is it?
Citric acid is a weak organic acid commonly used in the food, cosmetic and pharmaceutical industry. The parent base of citric acid, citrate, is a component of the Krebs cycle, and occurs naturally during metabolism in all living organisms. It is found naturally in citrus fruit such as lemons and limes and is used as a natural preservative. Monohydrate citric acid has one water molecule as part of it’s chemical formula, and exists as a white powder.
Citrate or citric acid is often used to adjust pH, to add sour flavor to foods and beverages, and to form the salt derivative of minerals and metals for pharmaceuticals, as in the case of potassium citrate, a dietary supplement. According to the FDA Select Committee on Generally Recognized as Safe (GRAS) food substances, citrate salts, including citric acid, are generally regarded as safe when used in normal quantities.
What is trisodium citrate dihydrate
Sodium citrate, (molecular formula: Na3C6H5O7 • 2H2O) has molecular weight of 294.1, is a colorless crystal or white crystalline powder product; it is odorless, salty taste, and cool.It will lose its crystal water at 150 °C and will be decomposed at even higher temperature. It also has slight deliquescence in wet air and has weathering property upon hot air. It is soluble in water and glycerol, but insoluble in alcohol and some other organic solvents. Sodium citrate has no toxic effect, and has pH adjusting capability as well as having a good stability, and therefore can be used in the food industry. Sodium citrate has the greatest demand when being used as a food additive; As food additives, it is mainly used as flavoring agents, buffers, emulsifiers, bulking agents, stabilizers and preservatives; in addition, combination between sodium citrate and citric acid can be used in a variety of jams, jelly, juice, drinks, cold drinks, dairy products and pastries gelling agents, flavoring agents and nutritional supplements.
- It can be used as Ph adjusting agents and emulsifying enhancers applied to jam, candy, jelly and ice cream; its combination with citric acid has an effect of alleviating tour; it also has effects on forming complex with metal ions. China rules that it can be applied to various types of food with appropriate usage according to the absolute necessity.
- It can be used as a food additive, as complex agent and buffering agent in electroplating industry; at the field of pharmaceutical industry, it is used for the manufacturing of anti-clotting drugs; and used as the detergent additives in light industry.
- It is used as the analysis agents used for chromatography analysis and can also used for preparing bacterial culture medium; moreover, it can also be applied into pharmaceutical industry.
- The product can be used for the flavoring processing of food, as stabilizers, buffers and deputy complex-forming agents in non-toxic electroplating industry; at pharmaceutical industry, it is used as anti-clotting agent, phlegm drugs and diuretics drugs. It can also be used in brewing, injection, newspaper and movies medicines.
It is produced by the neutralization of citric acid by sodium hydroxide or sodium bicarbonate. Dissolve sodium bicarbonate in water upon stirring and heating; add citric acid, continue to heat up to 85-90 °C; adjust the pH to 6.8; adjust active carbon for bleaching. Filter when the mixture is still hot; condense the filtrate under reduced pressure; cool and the crystal comes out; filter, wash, dry to obtain the final products of sodium citrate.
C6H8O7 + 3NaHCO3 → C6H5Na3O7 • 2H2O + 3CO2 ↑ + H2O
white powder or colourless crystals
Sodium citrate dihydrate consists of odorless, colorless, monoclinic crystals, or a white crystalline powder with a cooling, saline taste. It is slightly deliquescent in moist air, and in warm dry air it is efflorescent. Although most pharmacopeias specify that sodium citrate is the dihydrate, the USP 32 states that sodium citrate may be either the dihydrate or anhydrous material.
Sodium citrate is chiefly used as a food additive, usually for flavor or as a preservative.
Anticoagulant for collection of blood. In photography; as sequestering agent to remove trace metals; as emulsifier, acidulant and sequestrant in foods.
ChEBI: The dihydrate of trisodium citrate.
Sodium citrate, as either the dihydrate or anhydrous material, is widely used in pharmaceutical formulations.
It is used in food products, primarily to adjust the pH of solutions. It is also used as a sequestering agent. The anhydrous material is used in effervescent tablet formulations. Sodium citrate is additionally used as a blood anticoagulant either alone or in combination with other citrates such as disodium hydrogen citrate.
Therapeutically, sodium citrate is used to relieve the painful irritation caused by cystitis, and also to treat dehydration and acidosis due to diarrhea.
After ingestion, sodium citrate is absorbed and metabolized to bicarbonate. Although it is generally regarded as a nontoxic and nonirritant excipient, excessive consumption may cause gastrointestinal discomfort or diarrhea. Therapeutically, in adults, up to 15 g daily of sodium citrate dihydrate may be administered orally, in divided doses, as an aqueous solution to relieve the painful irritation caused by cystitis.
Citrates and citric acid enhance intestinal aluminum absorption in renal patients, which may lead to increased, harmful serum aluminum levels. It has therefore been suggested that patients with renal failure taking aluminum compounds to control phosphate absorption should not be prescribed citrate- or citric acid-containing products.
Sodium citrate dihydrate is a stable material. Aqueous solutions may be sterilized by autoclaving. On storage, aqueous solutions may cause the separation of small, solid particles from glass containers.
The bulk material should be stored in an airtight container in a cool, dry place.
Aqueous solutions are slightly alkaline and will react with acidic substances. Alkaloidal salts may be precipitated from their aqueous or hydro-alcohol solutions. Calcium and strontium salts will cause precipitation of the corresponding citrates. Other incompatibilities include bases, reducing agents, and oxidizing agents.
GRAS listed. Accepted for use as a food additive in Europe. Included in the FDA Inactive Ingredients Database (inhalations; injections; ophthalmic products; oral solutions, suspensions, syrups and tablets; nasal, otic, rectal, topical, transdermal, and vaginal preparations). Included in nonparenteral and parenteral medicines licensed in the UK. Included in the Canadian List of Acceptable Non-medicinal Ingredients.
What is ethanol,
Ethanol, also called ethyl alcohol, grain alcohol, or alcohol, a member of a class of organic compounds that are given the general name alcohols; its molecular formula is C2H5OH. Ethanol is an important industrial chemical; it is used as a solvent, in the synthesis of other organic chemicals, and as an additive to automotive gasoline (forming a mixture known as a gasohol). Ethanol is also the intoxicating ingredient of many alcoholic beverages such as beer, wine, and distilled spirits.
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There are two main processes for the manufacture of ethanol: the fermentation of carbohydrates (the method used for alcoholic beverages) and the hydration of ethylene. Fermentation involves the transformation of carbohydrates to ethanol by growing yeast cells. The chief raw materials fermented for the production of industrial alcohol are sugar crops such as beets and sugarcane and grain crops such as corn (maize). Hydration of ethylene is achieved by passing a mixture of ethylene and a large excess of steam at high temperature and pressure over an acidic catalyst.
Ethanol produced either by fermentation or by synthesis is obtained as a dilute aqueous solution and must be concentrated by fractional distillation. Direct distillation can yield at best the constant-boiling-point mixture containing 95.6 percent by weight of ethanol. Dehydration of the constant-boiling-point mixture yields anhydrous, or absolute, alcohol. Ethanol intended for industrial use is usually denatured (rendered unfit to drink), typically with methanol, benzene, or kerosene.
Pure ethanol is a colourless flammable liquid (boiling point 78.5 °C [173.3 °F]) with an agreeable ethereal odour and a burning taste. Ethanol is toxic, affecting the central nervous system. Moderate amounts relax the muscles and produce an apparent stimulating effect by depressing the inhibitory activities of the brain, but larger amounts impair coordination and judgment, finally producing coma and death. It is an addictive drug for some persons, leading to the disease alcoholism.
Ethanol is converted in the body first to acetaldehyde and then to carbon dioxide and water, at the rate of about half a fluid ounce, or 15 ml, per hour; this quantity corresponds to a dietary intake of about 100 calories.
Apigenin (AP) has many pharmacological activities. AP has poor solubility in some solvents. AP is insoluble in water and slightly soluble in ethanol (1.93 mg/ml). It has limited application and exploitation. Therefore, the liquid antisolvent precipitation (LAP) method was applied to improve the solubility of AP in ethanol by changing its crystal form or producing ultra-fine particles. Then, the inclusion complex of AP with 2-Hydroxypropyl-β-cyclodextrin (HP-β-CD) is prepared using the solvent removal method. The effects of various experimental parameters on the solubility of AP in ethanol were investigated through the single factor design. Under the optimum conditions, the AP-ethanol solution of 6.19 mg/ml was obtained. The inclusion complex of AP with HP-β-CD was obtained by the solvent removal method. The load efficiency (LE) and drug encapsulation efficiency (EE) of the inclusion complex of AP with HP-β-CD were 13.98%±0.14% and 97.86%±1.07%, respectively. SEM, FTIR, 1HNMR, XRD, DSC and TG were used to analyze the characteristics of the inclusion complex of AP with HP-β-CD. These results showed that the inclusion complex has significantly different characteristics with AP. In addition, the dissolution rate and solubility of the inclusion complex were approximately 15.24 and 68.7 times higher than AP in artificial gastric juice, and was separately 10.4 times and 40.05 times higher than AP in artificial intestinal juice. The bioavailability of inclusion complex increased 3.97 times compared with AP.
2-Hydroxypropyl-β-cyclodextrin Chemical Properties,Uses,Production
White to slightly yellow powder
Hydroxypropyl betadex occurs as a white or almost white, amorphous or crystalline powder.
2-Hydroxypropyl-β-cyclodextrin (HBC) is a widely used modified cyclodextrin, the lipophilic cavity formed by 7 glucose units. Drug solubility in water is greatly enhanced by complexing with 2-Hydroxypropyl-β-cyclodextrin.
enteric coating, sustained release formulations, buccal and transdermal drug delivery
2-Hydroxypropyl-β-cyclodextrin can be used as selective estrogen receptor modulator for the prevention of osteoporosis
Hydroxypropyl betadex is prepared by the treatment of an alkaline solution of b-cyclodextrin with propylene oxide. The substitution pattern can be influenced by varying the pH. Formation of O-6 and O-2 substituted products is favored by high and low alkali concentration, respectively. The mixture of products produced may be refined by preparative chromatography.
Hydroxypropyl betadex has been widely investigated in pharmaceutics and has principally been used as a solubilizer for hydrophobic molecules in oral liquids,oral solids, parenterals, pressurized metered dose inhalers, dry powder inhalers, and topical formulations. It has also been shown to act as a stabilizer during processing and storage of formulations.
Hydroxypropyl betadex inclusion complexes have been reported to show mechanical properties distinct from the pure materials. The reported advantage of hydroxypropyl betadex over unsubstituted b-cyclodextrin is its greater water solubility.
The pharmaceutical toxicology of hydroxypropyl betadex has been reviewed, and in general, the material was found to be of low toxicity. It has been suggested that hydroxypropyl betadex may have a synergistic toxic effect with, for example, carcinogens, by increasing their solubility and thus bioavailability.
Store in well-closed containers.
Included in oral and parenteral medicinal products. Included in an injectable preparation licensed in the UK for intramuscular or intravenous administration.
What is polysorbate 80
Polysorbate 80, also known as Tween 80, is a synthetic nonionic surfactant commonly used in food, cosmetics, and drug formulations as a solubilizer, stabilizer, or emulsifier [1–3]. Polysorbate 20 and 60 (Tween 20 and 60) are also included in this family of surfactants [1, 4]. It has also been used to prevent protein adsorption and/or aggregation . A wide range of pharmaceutical agents are available in formulations that contain polysorbate 80, including amiodarone , vitamin K , etoposide , docetaxel , various vaccines , protein biotherapeutics , erythropoietin-stimulating agents [9, 10], and fosaprepitant . Recent data have indicated that polysorbate 80 is a biologically and possibly pharmacologically active compound and consequently may alter the pharmacologic properties of the drug it is formulated with or may itself directly mediate adverse events [3, 12]. Consequently, polysorbate 80 has been implicated in some of the adverse reactions associated with drugs formulated with this vehicle.
This review covers the safety of polysorbate 80 in the oncology setting, focusing on polysorbate 80-associated adverse events that may have occurred with the use of docetaxel, darbepoetin alfa, epoetin alfa, and fosaprepitant.
This article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by any of the authors.
Overview of Polysorbate 80
Chemistry of Polysorbate 80
Polysorbate 80 is a synthetic surfactant composed of fatty acid esters of polyoxyethylene sorbitan [1, 2]. The fatty acid composition is primarily oleic acid, but other fatty acids, such as palmitic or linoleic acid, may be included (Fig. 1). Therefore, polysorbate 80 is usually available as a chemically diverse mixture of different fatty acid esters with the oleic acid comprising > 58% of the mix . However, the main component of polysorbate 80 is polyoxyethylene-20-sorbitan monooleate, structurally similar to polyethylene glycols. Polysorbate 80 has a molecular weight of 1309.7 Da and a density of 1.064 g/ml .
Polysorbate 80 has both hydrophobic and hydrophilic moieties [1, 2]. The hydrophobic moieties drive an interaction with the air-water interface or a solid-water interface, such as that found in vials, syringes, and other glass and plastic containers . The hydrophobic moieties of polysorbate 80 also result in the formation of micelles at concentrations above the critical micelle concentration of 0.01% (weight/volume) in protein-free aqueous solution . This formation of micelles may play a critical role in the mechanism of action of polysorbate 80. Enzyme-linked immunosorbent assays have shown that polysorbate 80 could activate the complement system, a multiprotein immune mechanism. Activating the complement system may lead to phagocytosis, stimulation, and recruitment of white blood cells, or perforation of plasma membranes, possibly leading to immunologic side effects such as acute hypersensitivity and systemic immune reactions . This possibility has been tested in a zebrafish model, where oxidized fatty acid residues in polysorbate 80 samples caused anaphylactoid reactions at the highest tested concentrations . Polysorbate 80 substituted for human serum albumin in an epoetin alfa preparation in Europe is thought to have played a role in the development of neutralizing antibodies and pure red blood cell aplasia . However, it is not yet known which specific parts of the chemical structure of polysorbate 80 are responsible for adverse events such as systemic and administration-site reactions.
Aqueous solutions of polysorbate 80, as well as the undiluted liquid, undergo auto-oxidation over time, with changes being catalyzed by light, increased temperature, and copper sulfate . Auto-oxidation leads to the formation of a variety of hydroperoxides, peroxides, and carbonyl compounds that may readily degrade proteins . During the initial stages of propagation, the peroxide formation is usually faster than its decomposition; eventually, the rates of formation and decomposition equalize, and then decomposition occurs faster than formation . Parameters such as surface tension and cloud point properties may be used to establish degradation in the hydrophilic chains .
Pharmacokinetic Properties of Polysorbate 80
In both animal  and clinical studies [18, 19], polysorbate 80 is rapidly removed from systemic circulation. The polysorbate 80 plasma concentration-time curve (AUC) in a patient administered an intravenous (IV) infusion of docetaxel 35 mg/m2 (polysorbate 80 1.75 g) showed a polysorbate 80 peak concentration of 304 μg/ml . The AUC for polysorbate 80 was 321.7 mg h/ml, with a short disposition half-life of 1.07 h and a total plasma clearance of 5.44 l/h. The volume of distribution of polysorbate 80 at steady state was similar to the total blood volume (4.16 l), suggesting that polysorbate 80 circulates as large micelles and does not significantly distribute outside the central compartment . In vitro studies suggest that polysorbate 80 is metabolized by rapid carboxylesterase-mediated hydrolysis .
Polysorbate 80 may potentially have an effect on the distribution and elimination of some IV-administered drugs with which it has been formulated (Table 1) [20–25]. This effect may lead to increased systemic exposure and decreased clearance of the drug . For example, polysorbate 80 may influence the binding of docetaxel in a concentration-dependent manner . A potential explanation of this effect may be that polysorbate 80 forms micellar complexes with proteins, so that the binding of docetaxel becomes saturated on single sites and the fraction of unbound drug increases [3, 25]. An alternative explanation is that the metabolism of polysorbate 80 and the subsequent displacement of oleic acid-mediated protein-binding sites may cause an increase in the fraction of unbound drug .
Polysorbate 80 and Adverse Events
Polysorbate 80 has been associated with a number of adverse events. In food, small concentrations of undigested polysorbate 80 may enhance bacterial translocation across intestinal epithelia, a potential explanation for an observed increase in the incidence of Crohn’s disease . In drug formulations, polysorbate 80 has been implicated in a number of systemic reactions (e.g., hypersensitivity, nonallergic anaphylaxis, rash) and injection- and infusion-site adverse events (ISAEs; e.g., pain, erythema, thrombophlebitis) [3, 35–37]. Polysorbate 80 has also been implicated in cases of renal and liver toxicity [38–40].
Description and Brand Names
Drug information provided by: IBM Micromedex
US Brand Name
- Sterile Saline Diluent Tip-Lok Syringe
Sodium chloride 23.4% injection is used to replenish lost water and salt in your body due to certain conditions (eg, hyponatremia or low salt syndrome). It is also used as an additive for total parenteral nutrition (TPN) and carbohydrate-containing IV fluids.
This medicine is to be given only by or under the supervision of your doctor.
This product is available in the following dosage forms:
In deciding to use a medicine, the risks of taking the medicine must be weighed against the good it will do. This is a decision you and your doctor will make. For this medicine, the following should be considered:
Tell your doctor if you have ever had any unusual or allergic reaction to this medicine or any other medicines. Also tell your health care professional if you have any other types of allergies, such as to foods, dyes, preservatives, or animals. For non-prescription products, read the label or package ingredients carefully.
Appropriate studies have not been performed on the relationship of age to the effects of sodium chloride 23.4% injection in the pediatric population. Safety and efficacy have not been established.
No information is available on the relationship of age to the effects of sodium chloride 23.4% injection in geriatric patients.
Studies in women suggest that this medication poses minimal risk to the infant when used during breastfeeding.
Although certain medicines should not be used together at all, in other cases two different medicines may be used together even if an interaction might occur. In these cases, your doctor may want to change the dose, or other precautions may be necessary. Tell your healthcare professional if you are taking any other prescription or nonprescription (over-the-counter [OTC]) medicine.
Certain medicines should not be used at or around the time of eating food or eating certain types of food since interactions may occur. Using alcohol or tobacco with certain medicines may also cause interactions to occur. Discuss with your healthcare professional the use of your medicine with food, alcohol, or tobacco.
Other Medical Problems
The presence of other medical problems may affect the use of this medicine. Make sure you tell your doctor if you have any other medical problems, especially:
- Bleeding problems or
- Congestive heart failure or
- Epilepsy (seizures) or
- Heart or blood vessel disease or
- Hypertension (high blood pressure) or
- Kidney disease, severe or
- Liver disease (eg, cirrhosis)—Use with caution. May make these conditions worse or increase the chance of side effects occurring.
- Fluid retention (swelling) or
- Hypernatremia (high sodium in the blood)—Should not be used in patients with these conditions.
- Kidney disease—Use with caution. Sodium chloride 23.4% contains aluminum, which may be toxic. The effects may be increased because of the slower removal of the medicine from the body.
A nurse or other trained health professional will give you this medicine in a medical facility. It is given through a needle placed into one of your veins after it has been diluted.
During the procedure, you will be awake and be asked questions about how you are doing by the health care team. This helps them to react quickly to any problems you might have and to keep side effects to a minimum.
It is very important that your doctor check your progress closely while you are receiving this medicine to make sure that the medicine is working properly and to check for unwanted effects.
This medicine may cause serious problems (eg, hypokalemia, acidosis, heart failure, pulmonary edema, or fluid retention) and are more likely to occur if sodium chloride is given for a long time or in high doses. Talk to your doctor if you have concerns.
Along with its needed effects, a medicine may cause some unwanted effects. Although not all of these side effects may occur, if they do occur they may need medical attention.
Check with your doctor immediately if any of the following side effects occur:
- Excessive blood loss
- burning pain in lower abdomen
- chest pain, severe
- feeling of heat
- feeling of warmth in the lips and tongue
- headache (severe or dull)
- loss of consciousness
- numbness of the fingertips
- pain in lower back, pelvis, or stomach
- ringing in the ears
- thirst (sudden) or salty taste
- vision problems
Incidence not known
- Bad smelling discharge from the vagina
- bleeding or redness at the injection site
- decrease in the amount of urine
- decreased urine output
- dilated neck veins
- extreme tiredness or weakness
- full or bloated feeling
- increase in bleeding from the uterus
- irregular breathing or heartbeat
- pain in the lower abdomen
- passing of pieces of tissue from the uterus
- pressure in the stomach
- stomach cramping
- swelling of the abdominal or stomach area
- swelling of the face, fingers, feet, or lower legs
- tightness in the chest
- troubled breathing
- weight gain
Get emergency help immediately if any of the following symptoms of overdose occur:
Symptoms of overdose
- usual tiredness or weakness
Some side effects may occur that usually do not need medical attention. These side effects may go away during treatment as your body adjusts to the medicine. Also, your health care professional may be able to tell you about ways to prevent or reduce some of these side effects. Check with your health care professional if any of the following side effects continue or are bothersome or if you have any questions about them:
Incidence not known
- Loss of appetite
- weight loss
Other side effects not listed may also occur in some patients. If you notice any other effects, check with your healthcare professional.
Call your doctor for medical advice about side effects. You may report side effects to the FDA at 1-800-FDA-1088.
Portions of this document last updated: Feb. 01, 2021
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