Growth factors and cytokines

The production, differentiation and survival of the various types of blood cell are tightly regulated by an interacting network of hormones, cytokines and growth factors. Species-specific activities of many of these chemical mediators, and their very low abundance, highlighted the need for biopharmaceutical products.

The most common uses of haemopoietic factors are for the treatment of various types of neutropenia, where specific white cell levels are depressed as a result of infection, immune disorders, recovery from chemotherapy or reaction to various drug regimens. They are especially useful in aiding recovery from the dose-limiting side effects of cancer chemotherapy. Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are used for this purpose to improve patient quality of life and allow the continuation of chemotherapy.

Erythropoietin (EPO), normally secreted by the kidney to stimulate the production of red blood cells, is the most successful biotechnology product so far marketed. EPO boosts red cell counts and reduces transfusion requirements for patients rendered anaemic by cancer chemotherapy or renal disease. Various forms of EPO with clearance profiles – and hence duration of action – modified by linkage with polyethylene glycol (PEGylation) or alteration of its glycosylation (see below and Chapter 17) are also available. Off-label use of EPO by athletes to improve performance has caused controversy.

Interferons are a complex group of proteins that augment immune effector cell function. Interferon-α was the first recombinant biotherapeutic agent approved by the FDA for cancer treatment. Recombinant interferons have been approved for melanoma, hepatitis C, Karposi’s sarcoma, T-cell lymphoma, chronic myelogenous leukaemia, multiple sclerosis and severe malignant osteopetrosis. Mechanism-based side effects have thus far restricted their utility to these severe disorders.

Hormones

Hormone replacement or augmentation is a commonly accepted medical practice in certain diseases of deficiency or misregulation. Insulin isolated from biological sources is administered to diabetics to control blood glucose levels. Immunological reaction to non-human (porcine or bovine) insulin preparations, which occur in a significant number of patients, are avoided by recombinant products incorporating parts of the human insulin sequence. A variety of human insulin and glucagon preparations are now on the market.

Human growth hormone (somatotropin) was originally developed for treating paediatric growth failure and Turner’s syndrome. Originally, growth hormone was extracted from human pituitary tissue post mortem, but this material carried a significant risk of transmitting Creutzfeld-Jakob disease, a fatal neurodegenerative condition now known to be transmitted by a prion, an abnormal protein found in affected nervous tissue, whose existence was unsuspected when human-derived growth hormone was introduced as a therapeutic agent. The production of human growth hormone by recombinant methods rather than extraction avoids this serious problem as well as providing a much more abundant source. Growth hormone has acquired notoriety since the potential for misuse to produce taller and stronger athletes was realized.

Human gonadotropin-releasing hormones have been extensively used in fertility management as well as in treating endometriosis and precocious puberty. Originally isolated from urine, there are now numerous recombinant products on the market.

Coagulation factors

Coagulation factors are a group of plasma proteins required for proper haemostasis. Deficiencies are associated with genetic lesions and occur as complications of viral infections such as hepatitis C and HIV. They were initially treated with plasma-derived concentrates, which carried a significant risk of viral and prion contamination. Recombinant factor VIII (Recombinate, Kogenate) and factor IX (BeneFix) avoid this risk and are now widely used.

Antithrombotic factors

To reduce blood coagulation, when conventional heparin or warfarin therapy is contraindicated, recombinant thrombin inhibitors (Leprudin, Bivalirudin) and antiplatelet gpIIb/IIIa antagonists (Eptifibatide) are now available. In conditions such as stroke, coronary thrombosis and pulmonary embolism early treatment with thrombolytic agents to relieve the vascular block is highly beneficial. Streptokinase and urokinase are proteases that process plasminogen to plasmin, activating its thrombolytic activity to dissolve fibrin clots. Newer products include tissue plasminogen activator (tPA) and TNKase, which catalyse the same reaction but in a fibrin-dependent fashion, so that plasmin production is concentrated in the region of the clot. These proteins are also less immunogenic than the bacterial streptokinase, which is important in cases where repeated administration of the thrombolytic agent is required.

Another regulator of coagulation is the serine protease protein C. The activated form of protein C breaks down the clotting factors Va and VIIIa and plasminogen activator inhibitor-1, tipping the balance in the favour of thrombolysis. These enzymes are important bio-pharmaceutical products that have no small-molecule counterpart.

Therapeutic antibodies

Antibody selection by phage display

Phage display technology (Benhar, 2001) is a useful way to identify antigen combining regions to produce monoclonal antibodies that bind to therapeutically relevant antigens. Bacteriophages (or phages) are viruses that replicate in bacteria, Escherichia coli being the organism of choice in most cases. For antibody selection (Figure 12.1) a large DNA library, encoding millions of different putative antigen-binding domains, is incorporated into phage DNA, so that each phage particle encodes a single antigen combining region. The mixed phage population is added to E. coli cultures, where the phage replicate, each phage particle expressing copies of a single antigen combining region on its surface. The phage suspension is applied to plates coated with the antigen of interest (‘panning’) and those phage particles expressing antigen combining regions recognizing the antigen stick to the plates. The adherent phages are isolated, allowing the antigen combining region-encoding DNA to be identified and inserted into the DNA sequence encoding an appropriate full-size antibody scaffold to produce a specific antibody, or into a reduced size, simplified monomeric framework to produce a single-chain (sFv) antibody.

Fig. 12.1 Antibody selection by phage display technique.

Therapeutic enzymes

Enzymes can be useful therapeutic agents as replacements for endogenous sources of activity that are deficient as a result of disease or genetic mutation. Because enzymes are large proteins they generally do not pass through cellular membranes, and do not penetrate into tissues from the bloodstream unless assisted by some delivery system. Lysosomal hydrolases such as cerebrosidase and glucosidase have targeting signals that allow them to be taken up by cells and delivered to the lysosome. Genetic lysosomal storage diseases (Gaucher’s, Tay–Sachs) are treated by enzyme replacement therapy. However, penetration of the enzymes into the nervous system, where the most severe effects of the disease are expressed, is poor. Cystic fibrosis, a genetic disorder characterized by deficits in salt secretion, is treated with digestive enzyme supplements and an inhaled DNase preparation (dornase-α) to reduce the extracellular viscosity of the mucous layer in the lung to ease breathing. All of these are produced as recombinant proteins.

Vaccines

Using the immune system to protect the body against certain organisms or conditions is a powerful way to provide long-term protection against disease. Unlike with small-molecule pharmaceuticals, which are administered when needed, once immunity is present subsequent exposure to the stimulus automatically activates the response. Most current vaccines are against disease-causing organisms such as bacteria, viruses and parasites. More complex conditions where the antigens are not so well defined are also being addressed by immunization. Vaccines are being tested for cancer, neurodegenerative diseases, contraception, heart disease, autoimmune diseases, and alcohol and drug addiction (Rousseau et al., 2001; Biaggi et al., 2002; BSI Vaccine Immunology Group, 2002; Kantak, 2003). Immune induction is complex. Pioneering experiments with attenuation of disease organisms showed that illness was not required for immunity. The goal in immunization is to retain enough of the disease-causing trait of an antigen to confer protection without causing the disease. For infectious agents, various methods of killing or weakening the organism by drying or exposure to inactivating agents still dominate manufacturing processes. Isolation of antigens from the organisms, or modifying their toxins, can be used in some cases. Vaccine production of isolated antigen ‘subunit’ vaccines benefits from protein engineering.

Novel approaches to presenting antigens are expected to have an impact on vaccination. An example is the phage display technique (Benhar, 2001), described earlier as a technique for antibody selection. The same approach can be used to provide the protein antigen in a display framework that enhances its immunogenicity and reduces the requirement for immune adjuvants (of which there are few approved for human use). Viruses encoding multiple antigens (‘vaccinomes’) can also be employed.

Genetic vaccination (Liu, 2003) employs a DNA plasmid containing the antigen-encoding gene, which is delivered to the host tissue by direct injection of DNA, or by more exotic techniques such as attaching the DNA to microparticles which are shot into tissues at high speed by a ‘gene gun’, or introduced by other transfection methods (Capecchi et al., 2004; Locher et al., 2004; Manoj et al., 2004). When the DNA is transcribed, the mRNA translated and the protein expressed in the host tissue, eukaryotic sequences will undergo appropriate post-translational modification, which does not occur with conventional protein vaccines. In general, partial sequences of pathogen-derived proteins are fully immunogenic, despite lacking the toxicity of full-length transcripts. The first human trial for a genetic vaccine against HIV took place in 1995, and others quickly followed, including hepatitis, influenza, melanoma, malaria, cytomegalovirus, non-Hodgkin’s lymphoma, and breast, prostate and colorectal tumours. Initial results have been promising. At the time of writing, recombinant vaccines against hepatitis A and B (e.g. Twinrix), papillomavirus virus for genital warts and cervical cancer (Gardisil), and against Haemophilus b/meningococcal protein for meningitis (Comvax) are approved. Single or multiple proteins from an organism can be included, and proteins that interfere with the immune response (common in many infectious agents) excluded.

Introduction of genetic material into cells

Nucleic acid polymers are large, highly charged poly-anions at physiologic pH. The mechanism by which such cumbersome hydrophilic molecules traverse multiple membrane lipid bilayers in cells is poorly understood, although a variety of empirical techniques for getting DNA into cells have been devised by molecular biologists. Eukaryotic cells use many strategies to distinguish between self and foreign DNA to which they are continually exposed. Some mechanisms apparently interfere with the incorporation and expression of engineered genes in gene therapy and other DNA transfer applications. Despite advances in technology, expression of foreign genes in cells or animals in the right place, at the right moment, in the right amount, for a long enough time remains difficult. Doing so in a clinical situation where so many more of the variables are uncontrollable is yet more challenging. The high expectations of gene therapies, which conceptually seemed so straightforward when the first trials were started, have run aground on the shoals of cellular genetic regulatory mechanisms. Effective gene therapy awaits the selective circumventing of these biological controls to deliver precise genetic corrections.

Delivery of nucleic acids

A potential gene therapy requires three major components: the payload to accomplish the mission, a targeting system to direct the vehicle and its payload to the correct body compartment in the correct cell population, and finally gene regulatory element(s), such as promoters/enhancers, to control the time and place of expression of the payload sequence(s). Table 12.2 compares the properties of some gene therapy vectors that have been used in clinical trials.

Table 12.2 Gene therapy vectors

To be clinically useful, a vehicle must elude host immunological or toxicological responses, must persist in the body, and must avoid sequestration in non-target organs such as the liver or kidney. Ideally, for clinical use one would want the ability to modulate or remove the genetic modification in case of adverse effects – the equivalent of discontinuing administration of a traditional drug. In practice, major difficulties are experienced in obtaining sufficient expression of the desired product for long enough to measure a positive clinical outcome.

Strategies for nucleic acid-mediated intervention

There are several points of attack for nucleic acid therapeutics. Most diseases are not due to an identified gene mutation, but rather reflect secondary cellular malfunction, often in response to events entirely external to the cell being targeted. Overexpressing proteins or fragments, either normal or mutated so as to compete with the normal protein for participation in cellular functions – known as the ‘dominant negative’ strategy – is a commonly used approach.

RNA metabolism can be modulated in many different ways, including:

• Antisense RNA

• Small interfering mRNA (siRNA)

• RNA decoys for viral RNA-binding proteins

• Specific mRNA-stabilizing proteins

• Interference with mRNA splicing to induce exon skipping or to correct abnormal splicing

• Sequence-specific cleavage by catalytic RNAs such as hammerhead and hairpin ribozymes. Bacterial introns that code for a multifunctional reverse transcriptase/RNA splicing/DNA endonuclease/integrase protein can be altered to target specific DNA sequences

• MicroRNA (miRNA) regulation.

Transcription of DNA into mRNA can also be controlled either through transcription factors or through antisense oligonucleotide triplex formation. Direct interference with genomic DNA through quadruplex formation is another strategy. These oligonucleotide sequences can be provided by biologically derived vector systems or by chemical synthesis. Short double- and single-strand oligonucleotide sequences are readily taken up by cells. Produced as single strands by automated solid-phase chemical synthesis, these short, single-stranded 8–20-nucleotide sequences can be chemically modified on the base, sugar, or phosphate linker to enhance their stability against nuclease degradation and cellular penetration (Figure 12.2).

Fig. 12.2 Chemical modification of nucleotides. Key for modifications: Sugar modifications: (a) fluoro-, (b) methoxy-, (c) methoxyethyl-, (d) propoxy-. Base modifications: (e) 5-methyl cytosine, (f) 5-propyne cytosine, (g) tricyclic cytosine. Phosphodiester backbone modifications: (h) phosphorothioate, (i) mopholino-, (j) methylene- (on PDF), (k) methylene-methylimino, (l) peptide nucleic acid (PNA).

Table 12.3 lists a number of clinical trials with nucleic acid-based therapeutics that are at different stages of completion.

Table 12.3 Clinical trials with antisense oligonucleotides

Manufacture of biopharmaceuticals

Expression systems

Full-size proteins, including a number of hormones, growth factors and mediators, are produced recombinantly by inserting the cDNA version of the mRNA sequence coding for the protein of interest into specially designed plasmid vectors that orchestrate the expression of the desired protein product in the cell type of choice (Figure 12.3). The characteristics of different expression systems are compared in Table 12.4.

Fig. 12.3 Generic vector for protein expression. The cDNA for the protein to be expressed is cloned into restriction sites in the multiple cloning site (MCS) of an expression vector which contains appropriate sequences for maintenance in the chosen host cell. Expression of the gene is controlled by inducible promoter. A ribosome-binding site (rbs) optimized for maximum expression directs translation, beginning at the ATG initiation codon. Either N-terminal or C-terminal tags (N-, C-Tag) can be used to aid in purification or stabilization of the protein, and a protease cut site may be used to remove the tag after purification. A translation terminator codon is followed (in eukaryotic cells) by a poly-A⁺ tail to stabilize the mRNA.

Table 12.4 Comparison of protein expression systems

These vectors contain a selection marker to maintain the vector in the host cell along with regulatory DNA sequences that direct the host cell transcriptional machinery to produce mRNA from the inserted cDNA sequence, with appropriate initiation and termination codons and a ribosome-binding site. A polyA⁺ tail is added to eukaryotic expression systems to stabilize the mRNA. mRNA transcription, and hence protein production, is controlled by an induction element, so that protein is produced only when required. This is important, as some expressed proteins are toxic to growing and dividing host cells. Other sequences, such as protein export signal peptides, hexahistidine tags, immunologic epitopes, or fusion with protein partners such as glutathione S-transferase, thioredoxin, maltose-binding protein or IgG Fc, facilitate secretion or provide handles for the detection/purification of the expressed protein. Purification tags are particularly useful in eukaryotic expression systems, which express exogenous proteins at lower levels than do bacterial systems, where the expressed protein can be 10–50% of the total cellular protein. The tags are designed to be removed from the protein once it has been purified.

Bacterial protein expression, usually in Escherichia coli, is the system of choice for high expression levels of many proteins, and because scale-up technology is well developed. However, not every protein is amenable to bacterial expression. Proteins that contain disulfide bonds or are comprised of multiple subunits are problematic, as are proteins containing critical carbohydrate, lipid or other post-translational modifications. In some cases the enzymes responsible for post-translational modification, such as fatty acylation, can be co-transfected with the desired protein. Highly expressed proteins are frequently produced as insoluble inclusion bodies in the bacterial cytoplasm or periplasmic space. Active proteins must be recovered from these inclusion bodies after denaturation and in vitro refolding. Intrinsic membrane-bound proteins can pose additional problems.

Eukaryotic microbial systems include Saccharomycetes cerevisiae and Picha pastoralis – yeasts that perform many mammalian post-translational modifications, including partial glycosylation. Insect cells, notably from Spodoptera frugiperda (Sf9), are infected with engineered baculovirus expression vectors carrying the protein of interest. This is a particularly efficient system for membrane protein expression. Many G-protein-coupled receptors, a large class of integral membrane proteins that includes many targets for currently marketed drugs, are often produced in these cells for use in screening assays for receptor ligands.

Cultured mammalian cells are used to express proteins on a research or production scale where faithful post-translational modification is required. Favourites include fibroblasts, anchorage-independent lymphocytes, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells. Mammalian cells grow more slowly and are more fastidious than the bacteria, yeasts or insect cells, and are therefore more expensive for production. Human cells are used in situations where there is some mammalian species dependence of bound carbohydrate. This can be particularly important for growth factors and the large glycoprotein hormones, as their glycosylation modulates the activity and stability of the protein product in vivo. Potential contaminating human pathogens in the human cell lines used for expression must be controlled. So must non-primate mammalian pathogens, as the possibility of transfer to humans cannot be ignored.

The use of transgenic animals and plants as ‘factories’ for genetically engineered proteins is discussed below.

Engineering proteins

Nature has devoted millions of years to optimizing protein structures for their biological roles. The natural configuration may not, however, be optimal for pharmaceutical applications. Recombinant DNA methods can be used to re-engineer the protein structure so as to better fulfil the requirements for a pharmaceutical agent.

Protein production

Although relatively small quantities of highly potent biologicals are normally required, production often presents problems. Only synthetic peptides and oligonucleotides and their derivatives can be produced by a scalable chemical synthesis. The others require large-scale fermentation or cell culture. The proteins and nucleic acids are then isolated and purified from the cells or media. Biotechnological process development is a complex field (Sofer and Zabriskie, 2000; Buckel, 2001; Gad, 2007). High-level production of recombinant proteins can result in heterogeneity because of the misincorporation of amino acids such as norvaline into the product, depending on the culture conditions, the nutrient composition of the medium and the producing organism. Post-translational modifications depend on the cultured organism and the subcellular localization of the protein. In cultured eukaryotic cells proteins undergo many kinds of post-translational modification, including glycosylation, sulfation, phosphorylation, lipidation, acetylation, γ-carboxylation and proteolytic processing. These and other modifications increase the heterogeneity of the product, affecting its biological half-life, immunogenicity and activity. The most homogeneous preparation consistent with retention of the important biological properties is standardized for production and a reproducible analytical profile is established. Nevertheless, protein products are invariably less homogeneous in composition than synthetic compounds, and the quality criteria for approval of clinical materials have to be adjusted to take account of this.

Formulation and storage conditions are critical because the protein primary amino acid sequences naturally display a variety of chemical instabilities. A major degradation route of protein products is through unfolding of the polypeptide chain and aggregation. Proteins have a finite half-life in an organism, governed partly by built-in chemical and conformational instabilities. Adsorption to surfaces of glass or plastic containers, particulates and air–liquid interfaces is often a cause of product loss.

The chemical reactivity of the various amino acid residues contributes to protein loss and heterogeneity. Succinimide formation, isomerization and racemization, as well as peptide bond cleavage, tend to occur at aspartate residues, to an extent that depends somewhat on the surrounding amino acid sequence. Examples include hACTH, soluble CD4, OKT-3 monoclonal antibody, hGH-releasing factor, IL-1β and hEGF. Oxidation, particularly at methionine residues, is catalysed by transition metal ions, pH, light, and various oxygen free radicals. Examples are relaxin A, hIGF-I and enkephalin analogs. Disulfide exchange (cysteine/cystine) can generate inappropriate folding or intermolecular bond formation, thereby promoting aggregation and precipitation of protein. Examples are interferon, and the acidic and basic forms of FGF. Beta-elimination at cystine, cysteine, serine and threonine leads to dehydroalanine formation and reaction with nucleophilic side chains. Anhydride formation between the α amino group on a protein and the C terminus is a major degradation pathway for insulin stored between pH3 and pH5. Sometimes peroxides contaminate the excipients and stabilizing agents, such as the polyethylene glycols or the surfactants Polysorbate 20 and Polysorbate 80 used in product formulation (see Chapter 16).

The first therapeutic proteins produced on an industrial scale were antibodies, which initially relied on immunization of large animals such as horses. Monoclonal technology paved the way for the production of murine antibodies in cell culture. With the ability to produce human antibody proteins and derivatives through molecular biological manipulation, the organism used to manufacture the protein is chosen on the basis of economics and requirements for post-translational modification. The characteristics of some commonly used organisms for protein production are summarized in Table 12.4.

Eukaryotic and prokaryotic microorganisms and eukaryotic cells in culture are the most common source of therapeutic proteins. Prokaryotic expression is generally the first choice, whereas higher organisms produce protein products that are most similar to human material and thus are more likely to be biologically active and pharmacokinetically stable. The production of proteins on a commercial scale by eukaryotic cell culture is, however, extremely expensive. The cells grow slowly (18–24 hours doubling time, compared with about 20 minutes for bacteria) and most require a serum source or expensive growth factor/hormone cocktails in the culture medium.

‘Pharming’ of protein expression in whole animals or plants is used for large-scale production of proteins for some therapeutics and for industrial applications. Transgenic farm animals – goats, cows, sheep and rabbits – have been engineered to secrete human proteins into their milk (van Berkel et al., 2002). Annual milk yields range from about 8 L (rabbit) to 8–10 000 L (cow). Expression levels of from 1 to 20 g of product per litre of milk have been achieved in favourable instances, allowing small herds of transgenic animals to do the work of large fermentation facilities. Many biopharmaceuticals are under development by specialized biotechnology companies (Table 12.5). The first drug produced in genetically engineered livestock (Atryn in goat milk) was approved by the FDA in February 2009. Thirty or more such products are expected to be registered in the next decade.

Table 12.5 Biopharmaceuticals produced in transgenic farm animals. (These products are in development; none have yet reached the market)

Plants can be directed to concentrate antibodies and other proteins in their leaves, seeds or fruit (Giddings et al., 2000; Daniell et al., 2001; Powledge, 2001; Streatfield and Howard, 2003), and are beginning to be used for the commercial production of antibodies and vaccines. Monoclonal antibodies expressed in plants – ‘plantibodies’ – have been produced in maize, tobacco and soybeans. Antibodies produced against Streptococcus mutans, the main cause of tooth decay in humans, have demonstrated efficacy against dental caries. CaroRX is in Phase II clinical trials for topical application for tooth decay. Other proteins, such as mouse and human interferon, human growth hormone, haemoglobin, human serum albumin and human epidermal growth factor, are also being targeted. Merispace, a recombinant mammalian gastric lipase, is being produced in corn for oral administration to counteract lipid maladsorption in cystic fibrosis and chronic pancreatitis Vaccine production for both animals and humans appears to be a most promising application of ‘pharming’. By splicing the gene for the antigen into a modified plant virus (cowpea mosaic virus), companies such as Agricultural Genetics (Cambridge, UK) have produced effective vaccines for animals against mink enterovirus, HIV-1 and foot and mouth disease, obtaining up to 200 doses of vaccine from a single cowpea (black-eyed pea) leaf. Other companies are pursuing vaccines for hepatitis A and B, cold and wart viruses and Plasmodium (malaria). Splicing the antigen genes directly into the plant genome has been successful for hepatitis B, Norwalk, cholera and rabies virus vaccines.

Feeding antigen-producing plants to animals has shown promise for inducing immune protection (Judge et al., 2004), despite the differences in the type of immune response from oral exposure and the standard humoral administration. The oral route would be ideal for human vaccines, particularly in countries where refrigeration is uncertain. Although antigen expressed in potato for use in humans was effective in animals, the cooking required for human consumption of the vegetable destroyed the immunogenicity of the antigen. Current efforts are developing the technology for human foods that are eaten raw and which are suitable for tropical climates, such as tomato and banana (Korban et al., 2002).

There are many developments in plant-derived biopharmaceuticals for human use, but none has yet become commercially available. Widespread environmental and other concerns among the general public about transgenic crop production are a significant impediment, especially outide of the United States.

Absorption of protein/peptide therapeutics

Elimination of protein/peptide therapeutics

Most therapeutic proteins are inactivated by proteolysis, producing short peptides and amino acids which then enter dietary pathways. Unlike small-molecule drugs, metabolites of protein therapeutics are not considered to be a safety issue. The rate and extent of degradation of protein therapeutics before excretion depend on the route of administration. Breakdown occurs in both plasma and tissues. Attempts to minimize these losses include the use of glycosylated proteins, which more closely resemble the native protein, or chemical modification of the protein with polyethylene glycol (PEG) chains or other entities. PEGylation can increase the plasma half-life of a protein between three- and several hundredfold. Besides reducing proteolysis, PEGylation can shield antigenic sites, enhance protein solubility and stability, and prevent rapid uptake by organs. With small peptides, cyclization or the inclusion of D-amino acid residues, particularly at the N terminus, can be used to protect against exoproteolysis, though this approach is of course applicable only to synthetic peptides and not to biopharmaceuticals.

The kidney is an important organ for the catabolism and elimination of small proteins and peptides. Proteins of 5–6 kDa, such as insulin, are able freely to pass the glomerular filter of the kidney. Passage through the glomerulus decreases to about 30% for proteins of 30 kDa, and to less than 1% for 69 kDa proteins. Some proteins, such as calcitonin, glucagon, insulin, growth hormone, oxytocin, vasopressin and lysozyme, are reabsorbed by the proximal tubules via luminal endocytosis, and then are hydrolysed in lysosomes. Linear peptides less than 10 amino acids long are hydrolysed by proteases on the surface of brush border membranes of the proximal tubules.

Liver metabolism is highly dependent on specific amino acid sequences in proteins. Cyclic peptides, small (<1.4 kDa) and hydrophobic peptides are taken up by carrier-mediated transport and degraded. Large proteins use energy-dependent carrier-mediated transport, including receptor-mediated endocytosis and transcytotic pathways (polymeric IgA), and are targeted to lysosomes.