Peng et al. Extensive clinical experience with pulmonary delivery of biotherapeutic insulin showed that patients with type 1 and type 2 diabetes switching from subcutaneous dosing resulted in larger ADA responses [ 75 , 76 ]. The antibodies were of the immunoglobulin G IgG class, were not neutralizing, and had no impact on clinical efficacy and safety. The importance of route of administration is not accepted by all; Schellekens argues that the immune reaction is not predicated by route of administration but rather is inherent in a therapeutic molecule itself [ 77 ].
Generally, short-term therapy is less likely to be immunogenic than long-term therapy, although intermittent treatment is more likely to elicit a response than continuous therapy [ 78 , 79 ]. Also, lower doses are generally more immunogenic than higher doses, as typically seen with mAbs where the phenotype is more tolerogenic.
This may be because of the evolution of the immune system to be generally less tolerant of low-abundance proteins. Therefore in clinical practice, high-dose regimens are used as a mode of therapy to induce tolerance e. The immune system can generate antibodies to therapeutic proteins by two general mechanisms: one relies on T cell costimulation of B cells while the other is independent of T cell [ 58 ].
Analysis of antibodies from clinical studies suggests that serious side effects are mainly driven by high levels of IgG antibodies, suggesting a T cell-dependent pathway. This protein is then internalized, processed, and returned to the surface as peptides bound to the MHC class II molecules. This in turn activates the B cells and leads to their differentiation into antibody-secreting short- and long-lived plasma B cells.
Some of these activated B cells also become memory cells, which maintain the pool of long-lived plasma cells and react rapidly to rechallenge by producing short-lived plasma cells. This T cell dependent immune response is thus usually long-lasting and of high titer, particularly for foreign or exogenous proteins [ 81 ]. In the T cell-independent antibody response, the ability to bypass Th cell costimulation leads to a more rapid antibody response. This type of response is typically evoked by particulate antigens and sequences of microbial and viral origin [ 6 ] repetitive epitopes termed pathogen associated molecular patterns.
Antigens that are expressed on the surface of pathogens in an organized, highly repetitive form can activate specific B cells by cross-linking of antigen receptors in a multivalent fashion [ 82 ].
This activation is dependent on the formation of a small number of antigen receptor clusters, each of which contains approximately 10 to 20 antigen-bound membrane Ig mIg molecules [ 82 ]. These clusters induce local membrane association of multiple activated Btk Bruton's tyrosine kinase molecules, which results in long-term mobilization of intracellular ionized calcium. Such persistent calcium fluxes efficiently recruit transcription factors, and thereby induce T-cell-independent B cell activation and proliferation. While this first signal of multivalent mIg cross-linking can induce B cell proliferation, a second signal in the form of engagement of members of the Toll-like receptor TLR family could selectively induce Ig secretion in B cells that were activated by multivalent, but not by bivalent, antigen receptor engagement.
Due to the lack of affinity maturation, this pathway typically results in an IgM-type response, which is transient, of low titer, and of poor specificity [ 72 ]. Changes to the structure of a therapeutic protein may alter its miscibility in ways that enhance aggregation or cause it to resemble a pathogen, thereby greatly increasing antigenicity. Typically, an immune reaction can be triggered by most therapeutic proteins inducing antibody responses.
Based on the trigger, the immune reaction can vary from low-titer, low-affinity, transient IgM antibody responses to high-titer, high-affinity responses, followed by class switching and IgG responses. Consequences of this transition can range from minimal to severe and life-threatening [ 72 ]. Most therapeutic proteins are synthesized in the endoplasmic reticulum ER and are eventually secreted. While some modifications occur before the proteins are secreted, others happen afterwards, during in vitro processing, including purification, formulation, and storage, and during administration into patients [ 83 ].
Modifications of proteins that occur in the ER, golgi, and exocellular spaces have been reviewed in detail by Fineberg et al.
- Post-translational modifications in the context of therapeutic proteins | Nature Biotechnology;
- Post Translational Modifications: what expression system to choose?.
- Post‐translational Modification of Protein Biopharmaceuticals | Wiley Online Books?
- Post-translational modifications of protein biopharmaceuticals.;
These modifications are disulphide bond formation, gamma carboxylation of glutamate residues, and beta hydroxylation of aspartate and asparagine residues in the ER; tyrosine sulfation, propeptide processing, O-linked glycosylation, phosphorylation, and amidation in the golgi; and deamidation, glycation, N-terminal pyroglutamate formation, oxidation, and proteolytic processing in the exocellular spaces.
Here, we will discuss protein structure, glycosylation, and chemical modifications. Posttranslational modifications can have direct or indirect effects on immunogenicity. The modified part of the biotherapeutic itself could induce an immune response, or its presence can affect the tertiary structure of the protein subtly causing the biotherapeutic to become immunogenic [ 4 ]. Primary amino acid sequence can affect protein structure, and hence immunogenicity, as is observed with animal-derived insulins [ 22 ]. For similar reasons, immunogenicity was higher for the first murine therapeutic antibodies, as compared to later chimeric, humanized, or fully human antibodies [ 84 ].
It is very interesting to note that while there are only 20 standard amino acids 19 amino acids and 1 imino acid , there are about different functional amino acids after hydrolysis. The role of PTMs is thus significant [ 83 ]. Over the years, a significant number of modifications have been identified and several of them characterized [ 3 , 85 ]. New epitopes in protein structure may be created due to the chemical modification of the protein, whereby new covalent crosslinks between amino acid residues are formed. These new protein structures could lead to the formation of aggregates, which may contain danger signals that greatly enhance immunogenicity.
Glycosylation is the covalent addition of carbohydrate molecules glycans to the protein surface. It is the most common, complex, and heterogeneous PTM that can occur in both endogenous and therapeutic proteins [ 3 , 86 ]. Almost half of the therapeutic proteins that are approved or in clinical trials are glycosylated [ 87 ]. The considerable heterogeneity in glycosylation profile of products can arise from the differences in the glycan itself type, structure or from the attachment pattern site, extent of occupancy of possible sites.
These variabilities may depend on the production and purification process [ 88 ]. Since glycans can influence the physicochemical e.
Glycosylation can have a direct or indirect impact on the immunogenicity of therapeutic proteins as well. The glycan structure itself can induce an immune response, or its presence can affect protein structure in such a way that the protein becomes immunogenic. Recent advances in analytical abilities, including matrix-assisted laser desorption ionization MALDI , electrospray ionisation mass spectrometry ESI-MS , and novel fluorescent tags for high performance liquid chromatography HPLC , can help in effectively characterizing and picking up potential changes in glycan profile of therapeutics [ 90 ].
Over the past decade, at least four nonhuman carbohydrate structures that are able to induce an immune response in humans have been identified. Most patients with hypersensitivity possess IgE antibodies against cetuximab before the start of therapy. Qian et al. Humans synthesize the sialic acid N-acetylneuraminic acid Neu5Ac but are not able to synthesize Neu5Gc [ 4 ]. Consumption of Neu5Gc-rich foods, for example, red meat and milk products, allows for the accumulation of Neu5Gc on the surface of epithelial and endothelial cells [ 93 ].
Injecting products that contain Neu5Gc in individuals with preexisting antibodies can cause the formation of immune complexes that potentially activate complement or affect half-life of the drug. Ghaderi et al. Maeda et al. However, these cells are capable of taking up these glycoforms from the cell culture media and metabolically incorporating them into the expressed protein [ 4 ]. Therefore, in addition to using these cell lines, media and other components should be void of components such as Neu5Gc [ 97 ].
Analysis of biotherapeutic mAbs purified from serum of subjects demonstrates that the PTM profile of the protein changes in vivo. Examples include deamidation at Asn and oxidation at Trp in the light chain and heavy chains, respectively, of two therapeutic mAbs [ 99 ]. Furthermore, a recent study shows that different levels of mannosylation of mAbs can have significant impact on pharmacokinetic parameters, including clearance and area under the curve AUC [ ]; however, the increase in mannose did not impact immunogenicity rates [ ].
Mannose receptors, expressed at high levels on DCs, mediate the capture, processing, and presenting of antigens mannose-expressing glycoproteins for an immune response. This response, depending on several factors, could either be immunogenic or tolerogenic [ , ].
Glycans may also indirectly impact the immunogenicity of biotherapeutics through changes in the folding, solubility, or stability of the proteins. Compared to glycosylation, other PTMs are less well understood [ , ]. A biopharmaceutical may be chemically modified through accidental degradation in one of the many bioprocessing steps: fermentation, virus inactivation, purification, polishing, formulation, filtration, filling, storage, transport, and administration. The susceptibility of an individual amino acid residue to chemical modification is dependent on neighboring residues; tertiary structure of the protein; and solution conditions such as temperature, pH, and ionic strength.
Chemical modification may give rise to a less favorable charge, thus leading to structural changes or even the formation of new covalent crosslinks [ ]. Covalent crosslinking could enhance immunogenicity by causing aggregation [ — ]. Multiple studies have indicated a strong correlation between aggregates and immunogenicity [ 89 , — ].
Deamidation, isomerization, and oxidation have also been associated with potential immunogenicity [ 4 ]. Deamidation of proteins accelerates at high temperature and high pH and can occur during bioprocessing and storage. Deamidation of Asn and Gln contributes to charge heterogeneity of therapeutic proteins, determines the irreversible thermal denaturation of proteins at acidic and neutral pH, regulates the rate of protein breakdown, and could shorten in vivo half-life.
Deamidation followed by isomerization of asparagine to isoaspartate isoAsp has been shown to alter protein structure, thereby potentially making the protein immunogenic [ ]. Deamidation can be accompanied by some degree of oxidation, conformational changes, and fragmentation and aggregation, again posing a serious risk of enhanced immunogenicity [ 4 ]. Oxidative chemical modification of amino acid residues alters secondary and tertiary protein structures. This favors interaction between protein surfaces and subsequently leads to noncovalent aggregation [ ].
Studies using metal-catalyzed oxidation MCO have shown that therapeutic proteins can aggregate and can also be immunogenic [ 4 , ]. Chemical stresses during manufacturing and storage can be caused by exposure to light or elevated temperatures and by the presence of oxygen, metal ions, or peroxide impurities from excipients. Trace amounts of iron, chromium, and nickel were found to leach into the formulation buffer via contact with the stainless steel surfaces typically used during bioprocessing [ ].
Tungsten oxide-mediated oxidation caused precipitation of monoclonal antibodies and was pH-dependent [ ]. Similarly in EPO, aggregation due to tungsten leachates from the container was associated with immunogenicity [ 54 ]. Despite limited information on the association of actual chemical modifications during biopharmaceutical manufacturing and immunogenicity, it is always prudent to be prepared for an untoward possibility.
Preventative measures should include careful evaluation of buffers, surface materials, and conditions during manufacturing, transport, and storage. Extensive characterization of molecules using techniques like size exclusion chromatography, supported by orthogonal techniques like analytical ultracentrifugation identifying aggregation [ ], circular dichroism CD , and intrinsic fluorescence spectroscopy, can indicate deviations from secondary and tertiary structures.
These steps incorporated into the process development will help in mitigating risks of immunogenicity. The ICH S6 Guideline preclinical safety evaluation of biotechnology-derived pharmaceuticals describes the need for detection and characterization of antibodies in repeat-dose studies using animal models. However, relevant species must be used for in vivo studies, that is, one in which the target epitope is expressed. Immune responses are species-specific; therefore, induction is not entirely predictive of antibody formation in humans [ , ].
Animal models are constrained by lack of genetic diversity which is a primary factor for diverse immune response frequently observed in human beings [ ].
Biosimilars and Their Structural Characterization
Rodent models for immunogenicity testing are, therefore, less useful than animals that show a higher degree of homology with humans and more genetic diversity than inbred mouse strains, such as nonhuman primates; however, these are not widely used due to ethical constraints. Conventional nontransgenic animal models can be useful for highly conserved proteins, but a lack of immune tolerance to human proteins limits their use for immunogenicity testing.
These animal models can be useful for comparing the immunogenicity of two similar products, that is, the immunogenicity of an originator and biosimilar product; this may not reflect the human situation but may provide a warning against advancement of a biosimilar if the immunogenicity profile observed differs from that of the originator. Despite the limitations associated with the use of animals to predict immunogenicity, several transgenic animal models have been generated for this purpose.
Transgenic mice are often the preferred in vivo model to predict immunogenicity as they are tolerant to the administered human protein [ , ] and can be used to study the immunogenicity of biotherapeutic aggregates. In a study by van Beers et al. In these experiments, immune tolerant mice were immunized with IFNb-1a formulations and antibody responses measured.
Only noncovalently bound aggregates that retained some native epitopes were able to break tolerance resulting in a transient immune response; removal of aggregates prevented this breakdown of tolerance [ ]. Additionally, mice expressing human MHC molecules can be used to compare antibody and T cell responses to vaccines and protein therapeutics [ ]. High ADA titers were observed after injection of a metal catalyzed, oxidized, and aggregated IgG1 sample in nontransgenic and transgenic mice [ 4 ].
Therapeutic interferons oxidized and aggregated via the same metal-catalysis method were able to overcome the immune tolerance of transgenic mice that were immune tolerant for the administered human proteins [ , ]. Use of animal models in immunogenicity testing is discussed more extensively in the review by Brinks et al. In vitro techniques can also be used to assess the immunogenic potential of therapeutic proteins.
These could be used to predict the risk of immunogenicity in preclinical setting. The expression of APC-surface molecules differs following activation; for example, the expression of MHC class I and II , costimulatory molecules, and cytokine receptors is enhanced. Flow cytometry is an in vitro technique that can be used to determine differences in cell surface molecule expression, indicative of APC maturation that may initiate T cell responses [ , ].
T cell proliferation assays are also useful tools to study the activation and proliferation of T cells in the presence of antigen [ ]. Additionally, the release of immunomodulatory cytokines can be characterized by enzyme-linked immunosorbent assay. T cells that respond to a particular epitope in vitro can be labeled with MHC class II oligomers and sorted by flow cytometry; the phenotype of responsive T cells can then be determined using intracellular cytokine staining [ , ]. These cytokines can be used as potential biomarkers for aggregate immunogenicity [ ].
It should be noted that these in vitro techniques may indicate the probability of an immune response for a biotherapeutic but cannot predict its clinical consequences. Correlative studies with marketed biotherapeutics in these assays may refine these methods further, to enable prediction of relevant immunogenicity [ 4 , ]. In addition to the assays described above, in silico techniques have been developed for the prediction of antigenicity by identification of potential T cell epitopes [ ]. In silico methods have been shown to successfully identify MHC class II-restricted epitopes within biotherapeutics [ ].
Knowledge of aggregation-prone regions may also help in the design and selection of biotherapeutic candidates and reduce aggregation concerns [ ]. For example, aggregation motifs that lack charge have been found in the light chain regions of mAbs, including Erbitux and Raptiva. This computational approach could, therefore, be useful to screen biotherapeutic candidates early in drug development [ ].
However, the challenge remains in identifying potential immunogenicity with low levels of aggregation induced naturally by PTMs as described previously especially in contexts of process change, shipping, and clinical use. The preclinical techniques to predict immunogenic potential described here are still exploratory.
Developing more robust methods to predict possible immunogenicity attributable to PTMs should be the way forward to reduce clinical risk. Prior to treatment, patients should be screened for established biomarkers to check for potential immunogenicity. A retrospective analysis of cetuximab evaluated whether the presence of pretreatment IgE antibodies against cetuximab is associated with severe infusion reactions SIRs during the initial cetuximab infusion.
This analysis used banked serum or plasma samples from cancer patients participating in clinical trials. Patients with a positive test indicating the presence of pretreatment antibodies had a higher risk of experiencing an SIR. Although this test had low positive predictive value, it clearly indicated an association between the presences of preexisting IgE antibodies against cetuximab with SIRs, supporting prior association studies [ ]. With adalimumab, dosing over the NAb response is probably effective in recapturing symptomatic response. In patients with Crohn's disease, adalimumab dose escalation is effective for recapturing symptomatic response after secondary loss of response, but more than half of the patients eventually experience a tertiary loss of response [ ].
An additional risk with dosing over the prescribed dose could involve adverse events such as serum sickness and hypersensitivity reactions [ ]. Another strategy commonly adopted with anti-TNF therapeutics is to switch the biologic when a patient becomes refractive to a particular anti-TNF.
Post-Translational Modification Analysis
In some cases, suppressing the immune response formation of ADAs with mild doses of methotrexate was seen to be beneficial [ ]. Figure 1 gives a schematic representation of managing immunogenicity. Management of immunogenicity in preclinical and clinical settings. In recent years, follow-on biologics or biosimilars and generic protein therapeutics have become more prevalent as the patents associated with the original drugs expire.
The first biosimilar reached the market almost a decade ago [ ]; and biosimilar use has been steadily rising.
Post-translational Modifications of Recombinant Proteins: Significance for Biopharmaceuticals
Managing immunogenicity arising due to biosimilars is another challenge. For small molecules approved in the EU, the generic paradigm applies; a product is pharmaceutically equivalent to a competitor molecule when it has the same qualitative and quantitative composition. If the products are shown through pharmacokinetic studies to have the same bioavailability, they are deemed bioequivalent. Generally, this is demonstrated in a limited number of studies in healthy volunteers [ ].
Once products are deemed bioequivalent, they are assumed to be therapeutically equivalent and essentially similar in terms of benefits and risks in vivo. However, such paradigm is not applicable for biopharmaceuticals. Biopharmaceuticals are large and intricate molecules and frequently subjected to extensive PTMs that are sensitive to differences in manufacturing conditions [ ]. Pharmaceutical equivalence for biopharmaceutical products cannot be directly demonstrated. Therefore, the biosimilar pathway was established. This exercise includes physicochemical studies, appropriate nonclinical studies, limited pharmacokinetic and pharmacodynamics studies, and comparative clinical studies to establish efficacy and safety European Medicines agency, London This stepwise approach starts with the assessment of critical quality attributes that are relevant to clinical outcomes in structural and functional characterization in manufacturing process of the proposed biosimilar product.
The FDA suggests that these critical quality attributes be identified first and then classified into three tiers depending upon their criticality: most Tier 1 , mild to moderate Tier 2 , and least Tier 3 relevant to clinical outcomes [ ]. However, even after demonstrating comparability, the products might not be similar in terms of risk of immunogenicity. Therefore, a detailed immunogenicity assessment is still warranted. Recent years have seen an expansion in the development and manufacturing of protein therapeutic drugs, both in terms of number of molecules and in terms of global production capacity.
In this review, we discussed the causes of immunogenicity which could be product-related inherent property of the product or might be picked up during the manufacturing process , patient-related, or linked to the route of administration. We also discussed the impact of PTMs of therapeutic proteins on immunogenicity; and it is clear that some PTMs lead to increased immunogenicity. Managing immunogenicity in both preclinical and clinical settings is very important. With the advent of novel analytical technologies, there has been a dramatic enhancement of the capability to analyze and characterize therapeutics.
Also, analysis of these proteins in vivo is critical to understand biological effects of PTMs. Relevant human immune system-specific animal models are now being established to study these biological effects. Future studies should focus on the development of sensitive diagnostics that can predict immunogenicity-mediated adverse events in small fraction of subjects that develop clinically relevant ADAs and hence mitigate the risk due to unwarranted immunogenicity.
The authors would like to acknowledge Dr.
This article has been cited by other articles in PMC. Abstract Today, potential immunogenicity can be better evaluated during the drug development process, and we have rational approaches to manage the clinical consequences of immunogenicity. Introduction Posttranslational modifications PTMs refer to enzymatic modifications that occur after translation, and which result in mature protein products. Immunogenicity and Its Causes Immunogenic response to therapeutic molecules can generate anti-drug antibodies ADAs , which can be either neutralizing or nonneutralizing.
Product- and Process-Related Causes of Immunogenicity The first therapeutic insulin products in the s were of bovine or porcine origin and were therefore immunogenic in humans. Table 1 Immunogenicity of FDA-approved biologics. Psoriasis 6. Open in a separate window. Mechanisms of Immunogenicity of Biotherapeutics The immune system can generate antibodies to therapeutic proteins by two general mechanisms: one relies on T cell costimulation of B cells while the other is independent of T cell [ 58 ].
T Cell-Dependent Immune Response Analysis of antibodies from clinical studies suggests that serious side effects are mainly driven by high levels of IgG antibodies, suggesting a T cell-dependent pathway. T Cell-Independent Immune Response In the T cell-independent antibody response, the ability to bypass Th cell costimulation leads to a more rapid antibody response.
Posttranslational Modifications and Their Correlation with Immunogenicity Most therapeutic proteins are synthesized in the endoplasmic reticulum ER and are eventually secreted. Protein Structure Primary amino acid sequence can affect protein structure, and hence immunogenicity, as is observed with animal-derived insulins [ 22 ]. Glycosylation Glycosylation is the covalent addition of carbohydrate molecules glycans to the protein surface. Managing Immunogenicity 5. Managing Immunogenicity in a Preclinical Setting The ICH S6 Guideline preclinical safety evaluation of biotechnology-derived pharmaceuticals describes the need for detection and characterization of antibodies in repeat-dose studies using animal models.
Managing Immunogenicity in the Clinic Prior to treatment, patients should be screened for established biomarkers to check for potential immunogenicity. Figure 1. Managing Immunogenicity against Biosimilars In recent years, follow-on biologics or biosimilars and generic protein therapeutics have become more prevalent as the patents associated with the original drugs expire. Conclusion Recent years have seen an expansion in the development and manufacturing of protein therapeutic drugs, both in terms of number of molecules and in terms of global production capacity.
Acknowledgments The authors would like to acknowledge Dr. References 1. Jensen O. Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry. Current Opinion in Chemical Biology. Burska A. Autoantibodies to posttranslational modifications in rheumatoid arthritis. Mediators of Inflammation. Jenkins N. Modifications of therapeutic proteins: challenges and prospects. Minimizing immunogenicity of biopharmaceuticals by controlling critical quality attributes of proteins.
Biotechnology Journal. Casadevall N. Antibodies against rHuEPO: native and recombinant. Nephrology Dialysis Transplantation. Mukovozov I. Factors that contribute to the immmunogenicity of therapeutic recombinant human proteins. Thrombosis and Haemostasis. Modifications Sorted by Name Concise description. Additional Reading. All All very common in eukaryotes and rare in prokaryotes. N-terminus Anywhere. One of the most common modifications. Amidation of alpha-CO2H occurs in secretory vesicles and granules.
All K. Cysteine disulfide bond formation occurs in the periplasmic space of bacteria and in the ER of eukaryotes. All All. Anywhere Anywhere. Needs to be followed by a G -. Possible regulator of protein-ligand and protein-protein interactions. N-terminus after cleavage of signal peptide.
Membrane tethering, reversible. Found in secreted proteins, involved in cell-cell recognition. All N, T, K. Hydroxylation of procollagen Pro and Lys residues occurs in the ER. Regulation of gene expression via histones methylation. Eukaryotes, Viruses Eukaryotes. Dar joined as post-doctorate in Prof. He is currently working as Sr. In addition to this, Dr. Dar is involved in structural and functional characterization of glycosylated therapeutic proteins from medicinal plants.
He has recently co-authored an edited volume book published by Springer, International Ltd. Laishram R. Singh is an Assistant Professor in the University of Delhi. During his Doctoral study he was engaged in investigating how small molecule compounds affect native protein structure, stability and enzymatic catalysis. At FCCC his main research interest encompasses understanding the Proteiostasis and modulators toward the functional restoration of mutant proteins including, mutants of p53, cystathionine beta synthase and methyl tetrahydrofolate reductase.
Currently Dr. Singh at Delhi University is working on understanding the protein covalent modifications by toxic metabolites present in the serum. Singh is also a well-known enzymologist and protein biochemist. He has published more than 40 publications in many esteemed journals in the field of proteiostatic regulation and protein modification by homocystein and other aldehydic compounds. Protein Research and Human Genetics, Dr.
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