Host cell protein

Last updated

Host cell proteins (HCPs) are process-related protein impurities that are produced by the host organism during biotherapeutic manufacturing and production. During the purification process, a majority of produced HCPs are removed from the final product (>99% of impurities removed). However, residual HCPs still remain in the final distributed pharmaceutical drug. Examples of HCPs that may remain in the desired pharmaceutical product include: monoclonal antibodies (mAbs), antibody-drug-conjugates (ADCs), therapeutic proteins, vaccines, and other protein-based biopharmaceuticals. [1] [2] [3]

Contents

HCPs may cause immunogenicity in individuals or reduce the potency, stability or overall effectiveness of a drug. National regulatory organisations, such as the FDA and EMA provide guidelines on acceptable levels of HCPs that may remain in pharmaceutical products before they are made available to the public. The accepted level of HCPs in a final product is evaluated on a case-by-case basis, and depends on multiple factors including: dose, frequency of drug administration, type of drug and severity of disease.

The acceptable range of HCPs in a final pharmaceutical product is large due to limitations with the detection and analytical methods that currently exist. [4] Analysis of HCPs is complex as the HCP mixture consists of a large variety of protein species, all of which are unique to the specific host organisms, and unrelated to the intended and desired recombinant protein. [5] Analysing these large varieties of protein species at very minute concentrations is difficult and requires extremely sensitive equipment which has not been fully developed yet. The reason that HCP levels need to be monitored is due to the uncertain effects they have on the body. At trace amounts, the effects of HCPs on patients are unknown and specific HCPs may affect protein stability and drug effectiveness, or cause immunogenicity in patients. [6] [7] If the stability of the drug is affected, durability of the active substance in the pharmaceutical product could decrease. The effects that the drug is intended to have on patients could also possibly be increased or decreased, leading to health complications that may arise. The degree of immunogenicity on a long-term basis is difficult, and almost impossible, to determine and consequences can include severe threats to the patient’s health. [5]

Safety risk

HCPs in biopharmaceutical products pose a potential safety risk to humans by introducing foreign proteins and biomolecules to the human immune system. Since common host cells used to produce biopharmaceutical drugs are E. coli , [8] yeast, [9] mouse myeloma cell line (NS0) [10] and Chinese hamster ovary (CHO), [11] the resultant HCPs are genetically different to what the human body [12] recognizes. As a consequence of this, the presence of HCPs in humans can activate an immune response, which can lead to possibly severe health concerns.

There is a correlation between the amount of foreign antigens (HPCs) in our body and the level of immune response our body produces. The more HCPs present in a drug, the higher the immune response that will be activated. Several studies have linked a reduction in HCPs to a decline in specific inflammatory cytokines. [5] Other HCPs may be very similar to a human protein and may induce an immune response with cross reactivity against the human protein or the drug substance protein. The exact consequences of HCPs for an individual patient is uncertain and difficult to determine with the current analytical methods used in biopharmaceutical production and analysis. [5]

Analysis

HCPs are identified during the manufacturing of biopharmaceuticals as part of the quality control process. [5]

During the production process several factors, including the genes of the host cell, the way of product expression and the purification steps, influence the final HCP composition and abundance. [5] Several studies report that HCPs are often co-purified along with the product itself by interacting with the recombinant protein. [6]

Enzyme linked immunosorbent assay (ELISA) is the predominant method for HCP analysis in pharmaceutical products due to its high sensitivity to proteins, which allows it to detect the low levels of HCPs in produced drugs. [4] Even though the developmental process requires an extended period of work and several tests with animal models, analysis of HCP content in the final product can be rapidly performed and interpreted. [1] Whilst ELISA possesses the sensitivity to undergo HCP analysis, several limitations are associated with the procedure. The HCP quantification relies mainly on the quantity and affinity of anti-HCP antibodies for detection of the HCP antigens. Anti-HCP antibody pools cannot cover the entire HCP population and weakly immunogenic proteins are impossible to detect, since equivalent antibodies are not generated in the process. [4]

In addition, methods such as the combination of mass spectrometry (MS) and liquid chromatography (LC-MS) have been developed to allow for more efficient and effective HCP analysis and purification. These methods are able to:

Recently, the MS method has been further improved through the method SWATH LC-MS. SWATH is a data independent acquisition (DIA) form of mass spectrometry, where the mass range is partitioned in small mass windows, which is then analysed with tandem MS (MS/MS). The key advantages are the reproducibility for both individual HCP identification and absolute quantification by applying internal protein standards. [14]

Despite the solid improvements of this method of protein analysis, there are also limitations, the main of which is that it requires a high level of expertise and advanced instrumentation to conduct the analysis. [13]

See also

Related Research Articles

<span class="mw-page-title-main">Antigen</span> Molecule triggering an immune response (antibody production) in the host

In immunology, an antigen (Ag) is a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response.

<span class="mw-page-title-main">Proteomics</span> Large-scale study of proteins

Proteomics is the large-scale study of proteins. Proteins are vital macromolecules of all living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In addition, other kinds of proteins include antibodies that protect an organism from infection, and hormones that send important signals throughout the body.

Glycomics is the comprehensive study of glycomes, including genetic, physiologic, pathologic, and other aspects. Glycomics "is the systematic study of all glycan structures of a given cell type or organism" and is a subset of glycobiology. The term glycomics is derived from the chemical prefix for sweetness or a sugar, "glyco-", and was formed to follow the omics naming convention established by genomics and proteomics.

<span class="mw-page-title-main">Glycoprotein</span> Protein with oligosaccharide modifications

Glycoproteins are proteins which contain oligosaccharide (sugar) chains covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. Secreted extracellular proteins are often glycosylated.

<span class="mw-page-title-main">Protein production</span> Biotechnological process

Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. This includes the transcription of the recombinant DNA to messenger RNA (mRNA), the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations.

<span class="mw-page-title-main">Glycome</span> Complete set of all sugars, free or bound, in an organism.

A glycome is the entire complement or complete set of all sugars, whether free or chemically bound in more complex molecules, of an organism. An alternative definition is the entirety of carbohydrates in a cell. The glycome may in fact be one of the most complex entities in nature. "Glycomics, analogous to genomics and proteomics, is the systematic study of all glycan structures of a given cell type or organism" and is a subset of glycobiology.

<span class="mw-page-title-main">Monoclonal antibody</span> Antibodies from clones of the same blood cell

A monoclonal antibody is an antibody produced from a cell lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell.

An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that binds to the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized are also epitopes.

<span class="mw-page-title-main">Chinese hamster ovary cell</span> Cell line

Chinese hamster ovary (CHO) cells are a family of immortalized cell lines derived from epithelial cells of the ovary of the Chinese hamster, often used in biological and medical research and commercially in the production of recombinant therapeutic proteins. They have found wide use in studies of genetics, toxicity screening, nutrition and gene expression, and particularly since the 1980s to express recombinant proteins. CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics.

<span class="mw-page-title-main">Recombinant DNA</span> DNA molecules formed by human agency at a molecular level generating novel DNA sequences

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

Pharming, a portmanteau of farming and pharmaceutical, refers to the use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants that would otherwise not express those genes, thus creating a genetically modified organism (GMO). Pharming is also known as molecular farming, molecular pharming, or biopharming.

A biopharmaceutical, also known as a biological medical product, or biologic, is any pharmaceutical drug product manufactured in, extracted from, or semisynthesized from biological sources. Different from totally synthesized pharmaceuticals, they include vaccines, whole blood, blood components, allergenics, somatic cells, gene therapies, tissues, recombinant therapeutic protein, and living medicines used in cell therapy. Biologics can be composed of sugars, proteins, nucleic acids, or complex combinations of these substances, or may be living cells or tissues. They are isolated from living sources—human, animal, plant, fungal, or microbial. They can be used in both human and animal medicine.

Immunogenicity is the ability of a foreign substance, such as an antigen, to provoke an immune response in the body of a human or other animal. It may be wanted or unwanted:

Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. The process of "humanization" is usually applied to monoclonal antibodies developed for administration to humans. Humanization can be necessary when the process of developing a specific antibody involves generation in a non-human immune system. The protein sequences of antibodies produced in this way are partially distinct from homologous antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human patients. The International Nonproprietary Names of humanized antibodies end in -zumab, as in omalizumab.

<span class="mw-page-title-main">Fusion protein</span> Protein created by joining other proteins into a single polypeptide

Fusion proteins or chimeric (kī-ˈmir-ik) proteins are proteins created through the joining of two or more genes that originally coded for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. Recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics. Chimeric or chimera usually designate hybrid proteins made of polypeptides having different functions or physico-chemical patterns. Chimeric mutant proteins occur naturally when a complex mutation, such as a chromosomal translocation, tandem duplication, or retrotransposition creates a novel coding sequence containing parts of the coding sequences from two different genes. Naturally occurring fusion proteins are commonly found in cancer cells, where they may function as oncoproteins. The bcr-abl fusion protein is a well-known example of an oncogenic fusion protein, and is considered to be the primary oncogenic driver of chronic myelogenous leukemia.

<span class="mw-page-title-main">PEGylation</span> Chemical reaction

PEGylation is the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol polymer chains to molecules and macrostructures, such as a drug, therapeutic protein or vesicle, which is then described as PEGylated. PEGylation affects the resulting derivatives or aggregates interactions, which typically slows down their coalescence and degradation as well as elimination in vivo.

Biomolecular engineering is the application of engineering principles and practices to the purposeful manipulation of molecules of biological origin. Biomolecular engineers integrate knowledge of biological processes with the core knowledge of chemical engineering in order to focus on molecular level solutions to issues and problems in the life sciences related to the environment, agriculture, energy, industry, food production, biotechnology and medicine.

A subunit vaccine is a vaccine that contains purified parts of the pathogen that are antigenic, or necessary to elicit a protective immune response. Subunit vaccine can be made from dissembled viral particles in cell culture or recombinant DNA expression, in which case it is a recombinant subunit vaccine.

Mass spectrometric immunoassay (MSIA) is a rapid method is used to detect and/ or quantify antigens and or antibody analytes. This method uses an analyte affinity isolation to extract targeted molecules and internal standards from biological fluid in preparation for matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). This method allows for "top down" and "bottom up" analysis. This sensitive method allows for a new and improved process for detecting multiple antigens and antibodies in a single assay. This assay is also capable of distinguishing mass shifted forms of the same molecule via a panantibody, as well as distinguish point mutations in proteins. Each specific form is detected uniquely based on their characteristic molecular mass. MSIA has dual specificity because of the antibody-antigen reaction coupled with the power of a mass spectrometer.

Recombinant antibodies are antibody fragments produced by using recombinant antibody coding genes. They mostly consist of a heavy and light chain of the variable region of immunoglobulin. Recombinant antibodies have many advantages in both medical and research applications, which make them a popular subject of exploration and new production against specific targets. The most commonly used form is the single chain variable fragment (scFv), which has shown the most promising traits exploitable in human medicine and research. In contrast to monoclonal antibodies produced by hybridoma technology, which may lose the capacity to produce the desired antibody over time or the antibody may undergo unwanted changes, which affect its functionality, recombinant antibodies produced in phage display maintain high standard of specificity and low immunogenicity.

References

  1. 1 2 "Tracking Host Cell Proteins During Biopharmaceutical Manufacturing: Advanced Methodologies to Ensure High Product Quality". www.americanpharmaceuticalreview.com. Retrieved 2018-10-02.
  2. ↑ Goey CH, Alhuthali S, Kontoravdi C (2018). "Host cell protein removal from biopharmaceutical preparations: Towards the implementation of quality by design". Biotechnology Advances. 36 (4): 1223–1237. doi:10.1016/j.biotechadv.2018.03.021. hdl: 10044/1/85952 . PMID   29654903. S2CID   4870812.
  3. ↑ Dimitrov DS (2012). "Therapeutic Proteins". Methods in Molecular Biology. Vol. 899. pp. 1–26. doi:10.1007/978-1-61779-921-1_1. ISBN   978-1-61779-920-4. ISSN   1940-6029. PMC   6988726 . PMID   22735943.
  4. 1 2 3 Zhu-Shimoni J, Yu C, Nishihara J, Wong RM, Gunawan F, Lin M, et al. (December 2014). "Host cell protein testing by ELISAs and the use of orthogonal methods". Biotechnology and Bioengineering. 111 (12): 2367–2379. doi: 10.1002/bit.25327 . PMID   24995961. S2CID   23923786.
  5. 1 2 3 4 5 6 Wang X, Hunter AK, Mozier NM (June 2009). "Host cell proteins in biologics development: Identification, quantitation and risk assessment". Biotechnology and Bioengineering. 103 (3): 446–458. doi: 10.1002/bit.22304 . PMID   19388135. S2CID   22707536.
  6. 1 2 3 Bracewell DG, Francis R, Smales CM (September 2015). "The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk-based management for their control". Biotechnology and Bioengineering. 112 (9): 1727–1737. doi:10.1002/bit.25628. PMC   4973824 . PMID   25998019.
  7. ↑ Guiochon G, Beaver LA (December 2011). "Separation science is the key to successful biopharmaceuticals". Journal of Chromatography A. 1218 (49): 8836–8858. doi:10.1016/j.chroma.2011.09.008. PMID   21982447.
  8. ↑ Blattner FR, Plunkett G, Bloch CA, Perna NT, Burland V, Riley M, et al. (September 1997). "The complete genome sequence of Escherichia coli K-12". Science. 277 (5331): 1453–1462. doi: 10.1126/science.277.5331.1453 . PMID   9278503.
  9. ↑ Zagulski M, Herbert CJ, Rytka J (1998). "Sequencing and functional analysis of the yeast genome". Acta Biochimica Polonica. 45 (3): 627–643. doi: 10.18388/abp.1998_4201 . PMID   9918489.
  10. ↑ Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF, Agarwal P, et al. (December 2002). "Initial sequencing and comparative analysis of the mouse genome". Nature. 420 (6915): 520–562. Bibcode:2002Natur.420..520W. doi: 10.1038/nature01262 . PMID   12466850.
  11. ↑ Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, Sodergren EJ, Scherer S, et al. (April 2004). "Genome sequence of the Brown Norway rat yields insights into mammalian evolution". Nature. 428 (6982): 493–521. Bibcode:2004Natur.428..493G. doi: 10.1038/nature02426 . PMID   15057822.
  12. ↑ "The Sequence of the Human Genome". Science. 291 (5507): 1155.4–1155. 2001-02-16. doi:10.1126/science.291.5507.1155d. ISSN   0036-8075. S2CID   196263909.
  13. 1 2 Pilely K, Johansen MR, Lund RR, Kofoed T, Jørgensen TK, Skriver L, et al. (January 2022). "Monitoring process-related impurities in biologics-host cell protein analysis". Analytical and Bioanalytical Chemistry. 414 (2): 747–758. doi:10.1007/s00216-021-03648-2. PMC   8483941 . PMID   34595561.
  14. ↑ Heissel S, Bunkenborg J, Kristiansen MP, Holmbjerg AF, Grimstrup M, Mørtz E, et al. (July 2018). "Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins: A case study of a bacterially expressed recombinant biopharmaceutical protein" (PDF). Protein Expression and Purification. 147: 69–77. doi:10.1016/j.pep.2018.03.002. PMID   29526817. S2CID   4741772.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.