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 A monoclonal antibody (mAb, more rarely called moAb) 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. Monoclonal antibodies ca ...

(mAbs), antibody-drug-conjugates (ADCs),

therapeutic protein A biopharmaceutical, also known as a biological medical product, or biologic, is any pharmaceutical drug product manufactured in, extracted from, or semisynthesis, semisynthesized from biology, biological sources. Different from total synthesis, ...

vaccine A vaccine is a biological Dosage form, preparation that provides active acquired immunity to a particular infectious disease, infectious or cancer, malignant disease. The safety and effectiveness of vaccines has been widely studied and verifie ...

s, and other protein-based

biopharmaceutical 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, t ...

s. HCPs may cause immunogenicity in individuals or reduce the potency, stability or overall effectiveness of a drug. National regulatory organisations, such as the

FDA The United States Food and Drug Administration (FDA or US FDA) is a federal agency of the Department of Health and Human Services. The FDA is responsible for protecting and promoting public health through the control and supervision of food ...

and EMA provide guidelines on acceptable levels of HCPs that may remain in pharmaceutical products before they are made available to the public. Currently, the acceptable level of HCPs in pharmaceutical drugs range from 1-100ppm (1–100 ng/mg product). However, 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. Analysis of HCPs is complex as the HCP mixture consists of a large variety of

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

species, all of which are unique to the specific host organisms, and unrelated to the intended and desired

recombinant protein Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be fou ...

. 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 Protein folding is the physical process by which a protein chain is translated to its native three-dimensional structure, typically a "folded" conformation by which the protein becomes biologically functional. Via an expeditious and reproduci ...

and drug effectiveness, or cause

immunogenicity 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: * Wanted immunogenicity typically relates to vaccines, where the injectio ...

in patients. If the stability of the drug is affected, durability of the

active substance An active ingredient is any ingredient that provides biologically active or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of the body of humans or animals. T ...

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.

Safety risk

HCPs in biopharmaceutical products pose a potential safety risk to humans by introducing foreign proteins and biomolecules to the human

immune system The immune system is a network of biological processes that protects an organism from diseases. It detects and responds to a wide variety of pathogens, from viruses to parasitic worms, as well as cancer cells and objects such as wood splinte ...

. Since common host cells used to produce biopharmaceutical drugs are ''

E. coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...

'',

yeast Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitut ...

, mouse myeloma cell line ( NS0) and Chinese hamster ovary (

CHO Cho or CHO may refer to: People * Chief Happiness Officer Surnames * Cho (Korean surname), one romanization of the common Korean surname * Zhuo (), romanized Cho in Wade–Giles, Chinese surname * Cho, a Minnan romanization of the Chinese sur ...

), the resultant HCPs are genetically different to what the

human body The human body is the structure of a Human, human being. It is composed of many different types of Cell (biology), cells that together create Tissue (biology), tissues and subsequently organ systems. They ensure homeostasis and the life, viabi ...

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

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

s (HPCs) in our body and the level of

immune response An immune response is a reaction which occurs within an organism for the purpose of defending against foreign invaders. These invaders include a wide variety of different microorganisms including viruses, bacteria, parasites, and fungi which could ...

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

cytokine Cytokines are a broad and loose category of small proteins (~5–25 kDa) important in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm. Cytokines have been shown to be involved in autocrin ...

s. 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.

Analysis

HCPs are identified during the manufacturing of biopharmaceuticals as part of the quality control process. During the

production process Industrial processes are procedures involving chemical, physical, electrical or mechanical steps to aid in the manufacturing of an item or items, usually carried out on a very large scale. Industrial processes are the key components of heavy ind ...

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. Several studies report that HCPs are often co-purified along with the product itself by interacting with the recombinant protein. Enzyme linked immunosorbent assay (

ELISA The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence ...

) 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. 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. 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 An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...

for detection of the HCP

s. 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. Methods such as the combination of

mass spectrometry Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...

(MS) and

liquid chromatography In chemical analysis, chromatography is a laboratory technique for the Separation process, separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it ...

( LC-MS) have recently been developed to allow for more efficient and effective HCP analysis and purification. These methods are able to: * Detect varying protein concentrations in a complex sample * Track an ever-changing HCP population and their concentrations during a manufacturing process * Analyse many proteins at once * Measure low abundant HCPs overshadowed by the high abundant target protein product 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 Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass spectrometry, mass analyzers are coupled together using an additional reaction step to increase their abilities to Analytical chem ...

). The key advantages are the reproducibility for both individual HCP identification and absolute quantification by applying internal protein standards. Despite the solid improvements of this method of protein analysis, there are also limitations, the main of which is to take into account the properties of the sample matter, thus: sampling conditions, limited technical variability, and the number of missing values in the sample - lead to infelicity that can be challenged in statistical analysis by factoring in batch effects, sampling effects, and the increase in missing identifications. {{cite web, title=Cell tissue proteome analysis, url=https://www.proteiq.com/services/cell-protein-analysis, website=Proteiq, publisher=ProteiQ Biosciences, access-date=22 July 2022

References

Biotechnology

Safety risk

Analysis

See also

References