See also CRISP gene editing, RNA interference and Gene Delivery. 

Companies: Dark Horse Consulting Group

Gene therapy encompasses the genetic modulation of cells and/or tissue in order to achieve a therapeutic effect. Gene therapy comes in a variety of forms from standard gene replacement strategies (for loss of function abnormalities), to gene silencing strategies (for gain of function abnormalities) and gene editing techniques. In all cases, the therapeutic genetic material is packaged in either a recombinant virus or non-viral vehicle and is delivered to cells via either in vivo or ex vivo routes of admistration. (Henckaerts, “What re the issues associated with developing gene therapies for rare disease and are the current development models working?” Cell & Gene Therapy Insights 2024: 10(5), 773-784)

Cell and gene therapy (CGT) products represent a diverse class of advanced therapies with the potential to treat and cure the underlying cause of a disease. Over the last several years the number of FDA approved CGTs has steadily increased form two products in 2015 to a total of 28 in 2024 (excluding cord blood dervived therapies, off-market and withdrawn licenses). These 38 approvals are spread across 3 major product classes: cell therapies, gene therapies and gene-modified cell therapies. The majority of approved products utilize viral vectors to deliver genetic material, with adeno-associated virus (AAV) being the predominant platform for in vivo gene therapies and lentivirus (LVV) being the predominant modality sued ex vivo for gene modified cell therapies. AAV delivers genetic material that forms episomal DNA in a cell which largely does not integrate into the host genome. This leads to stable expression in non-dividing cells and transient expression in dividing cells, as each cell division will ultimately dilute the episomes. In contrast, LVV delivers its genetic payload into both dividing and non-dividing cells leading to stable expression, and integration is typically random. (Christina Fuentes “Coming of age: an overview of the growing toolbox for gene editing and its use in CGT applications” Cell & Gene therapy insights, 2024; 10(9), 1221-1236).

Ex vivo Gene Therapies:

Ex vivo genome editing (GE) cell and gene therapy (CGTs) are the most advanced in development (i.e., CASGEVY commercial approval) and comprise the majority of GE CGTs in the clinical. In most cases, delivery is achieved via electroporation to the target cells ex vivo while in vivo GE CGTs utilize viral and nonviral delivery platforms. Ex vivo GE CGTs offer an advantage of greater control of editing, as the target cell population can be precisely selected, and analysis of any off-target edits can be performed prior to administration to the patient. (Christina Fuentes “Coming of age: an overview of the growing toolbox for gene editing and its use in CGT applications” Cell & Gene therapy insights, 2024; 10(9), 1221-1236).

Ex vivo gene therapies include adoptive cell strategies that enhance the anti-tumour activity of lymphocytes to target rare blood cancers such as Kymriah and genetically modified CD34+ hematopietic stem cells to treat non-cancer related RD such as Strimvelis, Casgevy and Lenmeldy.

HSCs are the target cell population:

HSCs are a good target cell population in gene therapy due to their ability to repopulate the patient’s body and remain in the system indefinitely. One approach involves extracting the cells from the patient, culturing them ex vivo, genetically modifying them, and re-infusing them into the patient. In the meantime, the patient can receive chemotherpay or a conditioning regimen to create space in the bone marrow for the genetically modified cells. (Charlotte Barker “Advancing ene therapies for beta-hemoglobinopathies with novel genome and epigenome editing tools” Cell & Geen Therapy Insights, 2024: 10(9), 1163-1171).

In vivo Gene Therapies:

In vivo gene therapy involves direct administration (either locally or systemically) of the vectorized therapeutic genetic material with the intention of directly transfecting or transducing cells in situ for therapeutic effect. Vector systems for in vivo gene therapy are selected based on their specific targeting capacity for the intended tissue and their safety profiles. Currently, recombinant AAV has attracted the most attention, with seven AAV based products arleady on the market, including ntoable therapies such as Luxturna (for Leber congenital amaurosis (LCA)) and Zolgensma (for spinal muscular atrophy (SMA)).  (Henckaerts, “What re the issues associated with developing gene therapies for rare disease and are the current development models working?” Cell & Gene Therapy Insights 2024: 10(5), 773-784)

 RNA Therapy

Therapeutic RNA refers to antisense oligonucleotides (ASOs), such as gapmers, which contain DNA nucleotides flanked by RNA, small interfering RNAs (siRNAs) or large RNAs, such as messenger RNA (mRNA). These RNA therapies act by targeting RNA or proteins, by encoding missing or defective proteins, or by mediating DNA or RNA editing. Irrespective of their therapeutic mechanism of action, the large size of some therapeutic RNAs, such as mRNAs, their anionic charge, and their susceptibility to RNases present in both the bloodstream and tissues make it difficult for therapeutic RNA to enter cells efficiently and function on its own. To overcome the barriers to safe and effective RNA delivery, scientists have developed both viral vector and non-viral delivery systems that protect the RNA from degradation, maximize delivery to on-target cells and minimize exposure to off target cells. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022).

Oligonucleotide drugs, such as ASOs and siRNAs, that utilize enzymes endogenous to eukaryotic cells, such as RNAse H1 or the RNA induced silencing complex (RISC), respectively, facilitate delivery by not requiring the delivery of large enzymes. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022).

Although different RNA payloads can have different biochemical mechanisms of action, all of them must avoid clearance by off-target organs, must access the correct tissue, must interact with the desired cell type in a complex tissue microenviorment, must be taken up by endocytosis, and must exit the endosome, without eliciting a deleterious immune response. Although small oligonucleotide RNA therapeutics, including ASOs, siRNAs, and ADAR oligonucleotides, can be modified using stable chemistries and delivered using conjugates, mRNA based and DNA based therapeutics require a vehicle for entry into the cell. To facilitate this process, scientifists have developed several RNA delivery systems using a range of materials, including polymers and LNPs. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)).

RNA Interference (RNAi): (See outline)

Companies: Alnylam Pharmaceuticals

RNAi is emerging as a strategy for treating a range of genetic disorders. Its therapeutic efficacy is based on gene silencing wherein a synthetic siRNA takes advantage of machinery in the cell to inhibit specific mRNA expression. Unlike genome editing which make changes to the cell’s DNA, the effect of RNAi is not permanent, providing an extra layer of safety in the clinic. The approach has been used to treat liver fibrosis where siRNA loaded lipid nanoparticles are used to deactivate hepatic stellate cells (HSCs). The siRNA maket is estimated to surpass 67 billion by 2036. (Nnenna Ohaka “Aptamer shares jump 23% following siRNA deal with AstraZeneca”, Tides Global, July 16, 2024).

siRNA interferes with mature mRNA, which is easier to acheive than nuclear delivery. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)

mRNA:

Another type of RNA therapeutic is mRNA, which can encode proteins that have therapeutic activity. Because of their size, mRNAs are in vitro transcribed and cannot currently be made with site specific chemical modifications using solid state synthesis. mRNA can be used to replace protein, using replacement therapies, to reduct protein levels, using Cas9 cutting approaches, or to repair protein mutations at the DNA level, using base editing. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)).

Rare Diseases and Gene Therapy:

RD is an umbrella term used to group a road range of individual diseases that share the common trait of having a low point prevalence within a population. There are an estimated 7k forms of RD. One of the key therapeutic modalities opted for the development of new treatments for RD is gene therapy. Despite the unmet medical needs and clear rationale for gene therapy in a great many different forms of RD, the number of gene therapy products available in the clinic remains relatively low. From a specific in vivo gene therapy perspective, hundreds of clinical trails utilizing AAV based gene therapy strategies have been undertaken for a variety of indications, yet the number of in vivo gene therapies on the market is still in single digits. The case for ex vivo gene therapies is arguably better, but the nature of these medicines often limits them to rare, oncology and immunodeficiency related indications, restricting the number of RD indications with which they are applicable. Currently, as little as 5% of all RDs have pharmacological intervention options, leaving the remaining 95% with no access to established drug based treatments. One of the reasons for this disparity can be attributed to the complex commercialization models associated with developing pharmaceutical agents that target small patient populations. another problem associated with RD drug development is the lack of characterization within the diseases themselves. Of the 7k odd RDs, only 355 of them have a code in the existing International Classifcation of Diseases (ICD).  (Henckaerts, “What are the issues associated with developing gene therapies for rare disease and are the current development models working?” Cell & Gene Therapy Insights 2024: 10(5), 773-784))

Ophan Drug Act (OdA): was created in an attempt to provide incentivization for the development of new drugs for RDs. The act deployed tax incentives, enhanced patent protection and marketing rights, and clinical research subsidies to spur the pharmaceutical industry into action.

Adenoassociated viruses (AAVs):

Companies:  MeiraGTx (optimization of AAV vectors and promoter sequences. Riboswitch technology that is designed for control of gene expression by oral small molecules).

3PBIOVIAN

Introduction:

AAVs is one of the most commonly used viral vectors for in vivo gene therapies. However, AAV based treatments often cause a short-term heptic immune response. This can be dampened with the use of antinflammatory medications such as steroids. A number of methods have been prosed to prevent immune detection of gene therapies, including the use of lipid nanoparticles for gene delivery or engineering viral capsids to evade recognition for patients’ antibodies. (Rsellini, BioProcess International, vlume 20, number 4, april 2022.

An AAV’s protein shell surrounds and protects its small (25 nm) single stranded DNA genome of about 4.7 Kb. That genome contains just three genes; rep (for replication), cap (for the capsid) and nap 9 for particle assembly). Lacking viral DNA, recombinant AAV (rAAV) is essentially a protein based nanoparticle engineered to pass through cell membranes, through which it carries and delivers a therapeutic DNA “cargo” into the cell’s nucleus. Because AAVs cannot replicate without outside help, they provide a safe vehicle to drive long term transgene expression after single infection. (BioProcess international, 21(1-2) 2023).

Applications:

–-Duchenne Muscular Dystrophy (DMD):

DMD is a rare genetic muscle disease. It occurs primarily in males, although females can be affected and they also pass the genetic mutation onto their children. DMD affects an estimated 12,000 to 15,000 people in the U.S. and 25,000 in Europe. DMD is the most common childhood-onset form of muscular dystrophy. Loss of strength and function typically first appear between 3 and 5 years of age. DMD results from a genetic mutation in the DMD gene on the X chromosome. This gene regulates the production of dystrophin, a protein essential to healthy muscle development and function. In people with DMD, dystrophin levels are absent or nearly absent, which causes permanent damage to muscle cells. See Dyne

—-Elevidy (delandistrogen moxeparvovec rokl): is an adeno-assocaited virus vector based gene theurapy of ambulatory pediatric patients age 4-12 with Duchenne muscular dystrophy with a confirmed mutation in the Duchenne muscular dystrophy gene. It is designed to deliver into the body a gene that leads to production of Elevidys micro-dystrophin that contains selected domains of the dystrophin protein present in normal muscle cells. The accelerated US Food and Drug Administration (FDA) approval of delandistrogene moxeparvovec was based on data from a randomized clinical trial that established that delandistrogene moxeparvovec increased the expression of the Elevidys micro-dystrophin protein observed in delandistrogene moxeparvovec-treated individuals aged four to five years with Duchenne muscular dystrophy.

–Spinal Muscular Atrophy:

SMA is a neurological disorder caused by a mutation in the SMN1 gene, which leads to a decrease in the SMN protein, a protein encessary for the survival of motor neurons.

—-Zogensma (Onasemnogene abeparvovec) (Novartis): is a gene therpay used to treat pediatric patients less than two years of age with spinal muscular atrophy with biollelic mutations in the survival motor nueron 1 gene. It involves a 1 time infusion of the drug into a vein. Zogensma is a biological drug which uses AAV9 viral capsids containing an SMN1 transgene.

–-REE65 Mediated Retinal Dystrophy:

Lentiviral Gene Therapies

Companies:  Avrobio  (AVRO: develops gene therapies to treat lysosomal storage diseases)

Lentiviral vecors are used mosly for ex vivo gene therpapy and are designed to deliver stable and durable integration of transgenes into a patient’s hematopoietic stem cells. The prcoess begins with harvesting CD34 stem cells form a patient. These cells are modified by using a lentiviral vector to insert a transgene that will enable production of a functional protein. The modified stem cells are infused back into the patient, where they engraft in the patient’s bond marrow. The cells divide to produce daughter cells, all carrying the transgene for the functional protein needed by those cells. In some cases, a protein is secreted to help other cells that have the defective gene, a process called cross-correction.

–Luxturna (voretigene neparvovec-rzyl): is an deno-assocaited virus vector based gene therpay approved for patients with confirmed biallelic RPE65 mutation-assocaited retinal dystrophy. Patients must have viable retinal cells as determiend by the treating physician(s). The gene therapy is not a cure for the condition, but substantially improves vision in those treated.

Non-viral vectors for gene delivery have attracted attention in the past decades becasue of their potential for limited immunogenicity, the ability to accomodate and deliver alrge size genetic materials and the potential for modification of theri surface structures.

Noevertheless, inherent to nonviral vectors are also important challenges. First, nonviral systems face aprpeciable plasmid loss during their mitotic segration in dividing cells. Second, silencing of transgene expression may occur due to epigentic events. Their, there is variable efficiency of plasmid delivery into various cells, both by physical and chemical methods. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

The major categories of non-viral vectors include cationic lipids and cationic polymers. 

Cationic lipid derived vectors:

Cationic lipid dervied vectors represent the most extensively investigated systems for non-viral gene delviery. Cationic polymer non-viral vectors have gained increasing attention becasue of flexibility in their synthesis and structural modifictions for specific biomedical applications. Both cationic lipid and cationic polymer systems deliver genes by forming condensed complxes with negatively charge deoxyribonucleic acid (DNA) through electrostatic interactions. Polyplexes formed between cationic polyemrs and DNA are relatively more stable than lipoplexes formed between cationic lipds and DNA. (Zhou “Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery” Nat Mater: 11(1) 82-90 (2012). 

Lipids and Lipid-based nanoparticles:

Companies: Beam Therapeutics

Polymers, liposomes, or other nanoscale structures can be modified to enhance their biocompatility, reduce cytotoxicity (e.g., PEG), and augment their potential to target specific tisseus or cell types. Polyemr and liposme ehcists increasingly collaborate with biologists to optimize the capablity of syntehtic nanostructures for improving nonviral gene delivery. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Concurrent advances in the development of syntehtic materials that encapsulate RNA, such as polymers, lipids and lipid nanoparticles (LNPs) ahve invigorated research into non-viral based delivery systems leading to FDA approval of subutaneously administered N-acetylgalactosamine (GalNAc)-siRNA conjugates that target hepatocytes, intravenously adminsitered LNP based siRNA drugs that target hepatocytes and emergency use authorizaton (EUA) and FDA approval for intramuscularly administered LNP based mRNA COVID vaccines. Tehse approvals suggest that improved delivery to non-liver tissues (also known as extrahepatic tissues) as well as local deliveyr to the CNS, eye and ear oculd result in new drugs. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022).

LNPs:

Lipid Nanoparticles (LNPs) are a key class of drug delivery system that includes nanoparticles approved by the FDA for liver siRNA delivery and form fRNA vaccine delivery. On the basis of the size of the hydrophoilic head group relative to the size of the hydrophobi tail or tails, lipids form distinct structures including micelles, liposomes and LNPs. FDA approved LNPs contain variations of four basic components: a cationic or ionizalbe lipid cholesterol, a helper lipid, and a poly (ethylene glycol) (PEG) lipid. In addition to the RNA payload, the Alnylam, Moderna and Pfizer/BioNTech/Acuitas LNPs include four components: the cationic or ionizable lipids DLin-MC3-DMA (Alnylam), SM-102 (Moerna) or ALC-0315 (Pfizer/BioNTech/Acuitas), cholesterol, the PEG-lipids PEG-2000-C-DMG (Alnylam), PEG-2000-DMG (Moderna) or ALC-0159 (Pfizer/BioNTech/Acuitas) adn DSPC. (Dahlman, “Drug delivery systems for RNA therapeutics” Nature Reviews Genetics, 23, May 2022)

LNPs are a key class of drug delivery system that includes nanoparticles approved by teh FDA for liver siRNA delivery and for mRNA vaccine delivery. On the basis of the size of the hydrophilic head group relative to the size of the hydrophobic tail or tails, lipids form distinct structures including micelles, liposomes and LNPs. FDA aprpoved LNPs contain variations of four basis components: a cationic or ionizable lipid, cholesterol, a helper lipid and poly(ehtylene glycol) (PeG)-lipd. Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)

Moderna discloses lipids and compounds useful for the nanopartcile composition for delivery of RNA therapeutics. (see US 9,533,047, US 9,867,888, US 10,195,156, 10,933,127  and US 10,799,463).

In addition to the RNA payload, the Alnylam, Moderna and Pfizer/tioNTech/Acuias LNPs comprise four componetns: the cationic or ionizable lipdis DLin-MC3-DMA (Alnylam), SM-102 (Moderna) or ALC-0315 (Pfizer), cholesterol, the PEG-lipids PEG-2000-C-DMG (Alnylam), PEG-2000-DMG (Moderna) or ALC-0159 )Pfizer). Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)

Liposomes and synthetic polymers are chemical means that exploit the nanoscale size and cotrollable surface properties of organic and polyemric molecuels for gene delivery. These chemical carreirs are intended ot match or exceed the performance of viral vectors with fewer immunogenic complciaitons. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

–PEGylated siRNA-loaded lipid nanopartciles: have addressed the greatest challenge in implementing siRNA therapeutics, which is their delivery. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

–In vivo Gene therapies:

In vivo gene therapeis can be delivered via the nonviral vehicle lipid nanoparticle (LNP). In fact, gene editing tools can be delivered to patients through intravenous infusion of LNPs targeted to hematopietic stem cells (HSCs), elimianting the need for transplantation altogtther. 

Virus-Like Particles:

Virus-like particles represent another class of nonviral gene dleivery vectors, and these typically consist of self assembled viral protein nanostructures. 

Polymers and Polymer-based nanoparticles:

Many non-viral RNA delivery systems utilize polymers and polymeric nanoparticles. Chemists can vary polymer traits including charge, degradability and molecualr weight, all of which influence how polymers delivery RNA into cells. One frequently used polymer is poly(lactic-co-glycolic acid) (PLGA). PLGA drug delivery systems have been approved by the FDA for the delivery of small molecule drugs but not for the devliery of nucleic acids. At neutral pH, PLGA does not have the positive charge required to complex the anionic RNA phosphodiester backbone. Thus, to utilze PLGA as an RNA deliveyr system, scientists have added cationic chemical groups such as chitosan to delivery siRNA in mice. (Dahlman, “Drug delivery systems for RNA therapeutics” Nature Reviews Genetics, 23, May 2022)

Episomal vectors/Episomes/Exosomes/Plasmids: 

Most plasmids (natural or aritifical) used in trasnfections, remain in the nucleus only transiently. Nonviral episomes have to pass thorugh a number of critical stages, starting with gaining cellular entry, until they can modulate sustained, long term maintenance within cells. The fate of pEPI-based, established, and onintegrating episomal vectors is characterized by a sequence of fundamental stages inside the host cell: establishment, replication, mitotic stability, and plasmid segregation in daughter cells at mitosis. These are ocmplex processes mediated by specific chromatin structures and DNA features, and studies during the recent years have greatly explanded how episomes can function as gene transfer vectors in a gene therapy context. Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Exosomes:

Exosomes are small secreted vesicles that are produced by all cells, are present at high concentrations in interstitial and other body fluids, and have been present throughout the life of every person and the evolution of every animal. They are in fact the only biologically nomral nanovesicle in existence. In contrast to LNPs, which have been shown to elicit cellular toxicity, exosomes were shown to have no adverse effects at any dose tested. Moreover, mRNA-loaded exomsomes were characterize by efficient mRNA encapsulation, high mRNA content, consistent and consistent size. (Gould, “Exosome-medaited mRNA delivery in vivo is safe adn can be used to induce SARS-CoV-2 immunity” J. Biiological Chemistry, 2021). 

Nonviral and nonintegrating episomal vectors are reemerging as a valid, alternative technology to integrating viral vectors for gene therapy, due to their more favorable saftey profile, significantly lower risk for insertional mutagenesis, and a lesser potential for innate immune reacitons, in addition to their low production cost. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Selaru (WO 2016/123556) discloses extracellular vesicles (Evs) derived from a cancer assocaited fibroblast (CAF) which includes a payload such as a polynucleotide such as miR-195, miR-126 or miR-192, identified as being dwon-regulated in the CAF. The extracellular vesicel selectively target a cancer cell. 

Episomes:

The term episome was propsoed by Fancois Jacob and Elie Wollman in 1958 to describe extrachromosomal genetic material that may replicate autonomously or become integrated into the chromosome. Although the term episome is now somewhat interchangeable with the term plasmid, episomes are larger in size and are retained for a longer period of tiem following transfection of host cells. Episome integration is a rare event and can occur through canonical sequence independent, nonhomolgous end joining or microhomology medaited end joining. Nevertheless. episomal integration must be inhibited if episomes are to become safe vihciles for gene therapy applciations. Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Episomal vectors or “episomes” are free, circular, extrachromosomal DNA molecuels of viral and nonviral origin. Viral epiosmes refer to viruses whose natural life cycle includes a stage of remaining as free, viral DNA within the cell nucleus, retaining the ability to encode proteins without integrating into the host cell’s genome. Examples include Adenoviruses (see outline). Non-viral episomes are effectively plasmids, usually of a larger size relative to conventional plasmids, and can exist independent of the genomic DNA for longer periods than covnentional plasmids. Both types of episomes are used as vectors of gene transfer in gene therpay. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Successful episome based gene expression hinges on the ability to safely and precisely deliver episome vectors to complex biological environments. Although bare nucleic acids can be delivered in vivo by direct introduction of DNA or RNA into cells, rpaid clearance and loss of expression limit the effectiveness of this approach. Several carreir systems can be utilized to deliver episomes and these include liposomes, syntetic polyemrs, or physical means of gene delivery (electroporation and sonoporation, among others). (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Episomes have also been successfully delivered in several tissues using lysin-PEG and polycationic comb polymers with nuclear loalizing sequences, among others. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

–Types of episomal vectors:

The most commonly used episomal vector ahs been pEPI-1, which originated form pGFP-C1 with the addition of the S/MARs anchoring elements, which keep the chromosomal DNA tethered into the nuclear matrix or scaffold. pEPI-1 was the first vecotr reproted as nonintegrative in the long term following trasnfection and the maintenance of key vecotr egiones. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Sleeping Beauty (SB) Transporson System:

The sleeping Beauty (SB) transposon system has emerged as a promising alternative to viral vectors. The SB system shares with viral vectors the need to integrate into the recipient cell’s genome to be functional. However, ocntrary to viral vectors, it does not show integration preference for sites within highly transcribed gehome domains. And since the vast majority of the genome does not contain highly transcribed gehnome domains, random integraiton of SB outside these domains reduces its risk for insertional mutagenesis. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

–Modified mRNA: Modern disclsoes a modified mRNA which overcome probleems with respect to the modulation of intracellular translation and processing of nuceic acids encoding polypetpides (US 9,950,068).

Companies:  Elektrofi (developed Hpercon which are protein microparticles. Gentle dehydration results in smooth, spherical microparticles that dissolve into fully functional protein monomers in the subcutaenous space. They take drug substances (typically in liquid form) and convert it through a series of proprietary methodologies into an ensemble of very smooth and spherical microphartciles. Then those particles are dispersed into an oily carrier medium that is eminetly flowable and syringable, even at very high concentraitons. Throughout the process, the protein molecule’s quality, shap and size are preserved and the concentraiton of injectable microparticle suspensions can ex eed 600 mg/mL. Not only are the formualtions capable of eliminating the need for infusions, they are capable of reducing the frequency of injections by as much as half thanks to the concentrated dosages). 

Brown (US 2007/0207210; see als WO 2005/112893) discloses methods of forming microparticles having a particle size of less than 200 microns, typically form 0.1 um to about 200 um which include an active agent such as an antibody. The particles are formed by added dissolving the active agent in an aqeuous solvent containing a “phase-separation enhancing agent (PSEA)” which induces or enhances the kiquid-solid phase seapration of the active agents from the solution when the solution is subjected tot he step of pahse seapration in which the active agent becomes solid or semi-solis. The PSEA reduces the solubility of the active agent when the solution is broght to the phase separation conditions such as lowering of temeprature. Examples of PSEAs are polymers such as PEG. The microparticels may also include one or more exicpients such as carohydrates, surfactants. 

Formation of Microparticles by Dehydration:

The dehydration of biologics is commonly employed to acheive solid-dose formulation and enhanced staiblity druing long-term preservation. (Needham “Microglassificaiton:  a novel technique for protein dehydration” Pharmaceutical Biotechnolgoy, 103:810-820, 2014). 

Bitterfield (US Patent Application No: 18/058306, published as US 2023/0093954) discloses isolation of enzymes (e.g., lysozyme) and other proteins using dehydration compositions such as pentanol. The dehydration method, referred to as “microglassification” produced particels of lysozyme. 

Needham (US 8,512,754) discloses a composition taht incldues glassified, staiblized particles having a low water activity (between aobut 0.1-0.9) which include an active agent such as a protein/antibody. The particles are produced by preparing a first aqueous phase that include an aqueous solution of the material of interest and a second pahse that incldues dispersing the aqueous phase into a non-aqeuous suspending solvent (deconaol, do-decanol, pentanol, etc) for forming the particles. 

Needham *”Microglassification:  a novel technique for protein dehydration” Pharmaceutical Biotechnolgoy, 103:810-820, 2014) dislsoes a process called “micrograssificaiton” that can rapidly and controllable dehydrate protein solutions into solid amorphous micrsophreres at room temepratures. Bovine serum albumin (BSA) microdroplets were suspended in pentanol or decanol using micropipietes. 

Needham and DeAngelo (US 8,013,022) also discloses using a two-phase micro system of glassificaiton; a frist phase is provided where the protein is prepared so as to form a micro droplet which is then disssolved into a second phase solvent (decanol, etc.). The method creates micro glass beads of a protein in a reduced hydration state, but not one that dehydrates the protin to such an extent taht it looses it ability to function upon reconstiution. 

Petrel (US 9,643,996) disclsoes producing microparticles of between 0.5-300 um that contain a high loading of bioactive macromolecuels such as protiens/antibodies. The processes combine atomization with solvent-assested dehydration (n-decanol; ethanol, pentanes, hexanes, ethers etc. ) to obtain solid particles that retain the biological activity of the payload. Initially, the protein solution is formed. The protein solution is then atomized to form liquid droplets such as by using an ultrasonic flow-through transducer. The liquid droplets formed by the atomization are then collected in a mixed dehydration solvent which dehydrates the protein by absorbing the water, resulting in the generation of protein microparticles. This is known as solvent-assisted dehydration. The prtoein microparticles are then separated form the dehydration solvent as for example by centrifugation, filtration, etc. 

Diseases of the CNS remain the world’s leading cause of disabilities, accounting for more hospitalization and prolonged care than almost all other diseases combined. Scuh CNS diseases include brain tumours, HIV encephaopathy, epielpsy, verebrovascular diseases and neurodegnerative disorders. The incidence of CNS disorders in human increases with age. Being the most delicate organ of the body, the brain is protected against potentially toxic substances by the blood-brain barrier (BBB); however, the BBB also prevents the penetraiton of most moleules that have potential activity in the CNS. 98% of small molecules are not able to cross the BBB and 100% of large molecuels cannot cross the BBB. (Amin “Getting into the brain” CNS Drugs, 2009, 35-58),

The highly restrictive nature of the BBB results in the majority of small polar molecules being incapable of dif- fusing into the brain (Figure 3C). These molecules include glucose, amino acids, nucleosides and many other mol- ecules. Given the essential roles these molecules play in cell growth and metabolism, it is essential for them to be transported across the BBB. This is achieved through the presence of carrier mediated transport proteins pre- sent on both the luminal and abluminal membranes of the endothelial cells of the BBB. (Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).

Receptor-Mediated Transport:

Receptor-mediated transport (RMT) is how large endogenous molecules, such as neuropeptides (insulin, transferrin or leptin) corrs the BBB. Insulin uptake is for example mediated by the insulin receptor, transferrin uptake is mediated by the transferrin receptor (Tfr), insulin-like growth factor (IGF) upstake is medaited by the IGF recptor and leptin uptake is medated by the leptin receptor. Amin “Getting into the brain” CNS Drugs, 2009, 35-58),

Designing genetically engineered fusion proteins to make use of the RMT to transport molecuels across the BBB is one approach for brain targeting. These proteins are prepared by fusing human transferrin to mouse-human chimeric IgG3 at 3 positons. Amin “Getting into the brain” CNS Drugs, 2009, 35-58),

The human insulin receptor (HIR), the most potent molecular trjan hourse in the BBB, is active in humans and rehesus monkeys. Brain drug delivery in the rhesus monkey was acheived by attaching a murine 83-14 mAb to the HIR. Amin “Getting into the brain” CNS Drugs, 2009, 35-58),

Fusion constructs:

Stah. (“Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs” Molecular Therapy) disclsoes enablement of self-devliery of the Cas9 ribo nuclear prtoeins. Four repeats of the positively charged Simian vacuolating virus 40 nuclear localizaiton sequences (SV40-NLS) were fused to the N temrinus along with two repeats to the C terminus of Cas9. Using a single guid to turn on the tdTomato reported form the lox-stlop-lox Ai9 mouse, they reproted edited striatal volcume of about 1.5 nm.

Aptamers as Drug Delivery Vehicles:

As aptamers function by molecular recognition, they can be developed for therapeutic applications with the same intended function as antibodies, such as drug delivery vehicles. Though analogous to their protein counterparts in regards to target recognition and application, aptamers possess numerous key advantages over antibodies and antibody fragments, the main being their size, pro- duction process and cost, stability and nucleic acid structure Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).

Aptamers have been covalently or physically functionalized with therapeutic compounds including chemotherapeutics, toxins, and therapeutic functionalized with therapeutic compounds including chemotherapeutics, toxins, and therapeutic oligonucleotides (siRNAs, miRNAs or antimiRs) and many methods of aptamer functionalization oligonucleotides (siRNAs, miRNAs or antimiRs) and many methods of aptamer functionalization have been proposed as valid means to improve the specificity of nanoparticles. These approaches have been proposed as valid means to improve the specificity of nanoparticles. Franciscis “Aptamer-Mediated Targeted Delivery of Therapeutics: An Update” Pharmaceuticals, 2016)

Antibody-Drug Conjugates (ADCs) are designed to bind to cell surface receptors and get internalized thorugh receptor medaited endocytosis, then trafficked into endosomes and ultimately lysosomes. Following internalization, ADC antigen complexes are fused with endosomes, which break them up for receptor recyling and transport ADs to lysosomes. Finally, an ADC molecule undergoes lysosomal degradation through a number of pathways to release its cytotoxin, which then binds to its target. (Chamow “Manufacturing challenges of therapeutic antibody -drug conugates, BioProcess Internaitonal, September 2023).

Preparation of Antibody-Cytotoxin Conjugates

In general, antibody cytotoxin conjugates are prepared by a multi-step process. First, antibody is covalently attached to a linker in a modification reaction and unreacted components and reaction products separated from antibody linker conjugate by desalting. Next, purified antibody-linker conjugate is reacted with modified cytotoxin to form the antibody-cytotoxin conjugate. The conjugate is purified from unreacted components, solvent and reaction products as by, for example, size exclusion chromatograph. For more information on purification of antibodies see antibody purification. Mazzola (US 2005/0031627)

The majority of ADCs are built on IgG1 scafolds. These complex about 150 kDa biomolecuels contain mltiple native sites for connjugation and can be modified to include additional reactive sites. (Own “Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry” AAPS Jouranl, 17(2), 2015). 

Dai (US 2011/0166319) discloses a prcoess for preapring an atnibody chemically coupled to a drug which cindlues the steps of frist covalently attaching a linker ot the antibody, an optional purificaiton step, conjugated a drug to the antibody-linker and a subsequent purificaiton step, and optional holding steps. 

Kaiser (US Patent Application No: 16/631,759, published as US 2020/0164338) discloses a method of prdoucing an antibody drug conjugation which includes the steps of pumpinng an antibody solution and a linker solution into a mixing device and then into a residence time device, followed along with a connection to the next unit operation, followed by conjugation of the drug. 

Chemical Modifications & Linking Sites on Antibodies:

Chemical modificiations for the functionalization of antibodies include modification with an ezyme such as alkaline phosphatase or peroxidase (HRP), iodation or addition of a chelating compound for radioisotopes and modification with a low molecular compound such as biotin. These modification are typically performed via lysine amino group, a cystein thiol group and an activated carboxyl group. These modificaitons are specific for the functional groups, but are not site-specific. Thus, the problems include reduction in the activity of antibodies due to the modification or the like of the antigen-bnding sties of the antibodies, and difficult control of the number of compounds to be bound. For ADCs, anticancer agents are bound to antibodies in a site-nonspecific manner. In order to overcome these problems, antibody modificaiton has been practiced using antibodies haivng a particular site specificaly introduced functional group. For example, a particular non natural amino acid or free cysten can be introduced by genetic manipulation. Also, a glutamine can be intorduced using transglutaminase (TG). (Ito US 2018/0141976). 

Antibody-drug conjugates (ADCs) are a subclass of biotherapeutics designed to faicilitate the targeted delivery of potent cytotoxic drugs to cancer cells. Depending on the conjugation chemistry, different types of ADCs can be constructed such as cystein-conjugated, lysine conjugated or site-specific ADCs. At a molecular level, all ADC molecuels bear complex chemical structures, combining the molecular characteristics of small molecule drugs with those of large molecule mAbs. In addition, the conjugation reaciton employed increases the complexity of ADC samples by producing a mixture of ADC molecules that are heterogeneous with respect to different drug to antibody ratios (DARs) and also the fact that conjugation is typically a random process becasue there are many surface accessible lysine residues. (Chen, “in-depth stuctural characterization of Kadcyla (ado-trastuzumab emtansine) and its biosimilar candidate” MABS 2016, 8(7), 1210-1223).

Although many drugs are amenable to direct conjugation to an antibody scaffold, heterobifunctional linkers often facilitate ADC bioconjugation. Linkers provide a functional handle for efficient conjugation to antibodies. Reactive handles often fetured in linker chemistry are grouped based on teh site of conjugation. N-hydroxysuccinimide esters are the most common choice for functionalizing amines, especially when coupling to E-lysine residues. For conjugation to cysteines, thiol-reactive maleimide is the most applied handle, alhtough it is also possible to create a disulfide bridge by oxidaiton with a linker bearing a sulfhydrl group. Aldehyde or keto functional groups such as oxidized sugar groups or pAcPhe unnatural amino acids can be reacted with hydrazides and alkoxyamines to yield acid-labile hydrozones or oxime bonds. In addition, hydrozine can be couple with an aldehyde via HIPS ligation to generate a stable C-C linkage. The mechanism of drug release is an important consideration in linker selection. Non-cleavable linkers rely on degradation of the scaffold within the lysosome after internalization. Alternatively, cleavage linkers respond to physicologic stimuli such as low pH, high clutathione concentrations and proteolytic clevage. Several non-clevable alkyl and polymeric linkers have been explored in ADC development. Non-clevable linkers require mAb degradation within the lysosome after ADC internalization to release active drug. Clevage linkers are popular in the ADC clinical pipeline with acid sensitive linkers such as hydrazones and silyl ethers. Hydrozones are easily synthesized and have a plasma half-life of 183 h at pH 7 and 4.4 h at pH 5, suggesting that they are selectively clevable under acidic conditions such as those found in the lysosome. Enzymatically clevage linkers are gaining significant attention due to superior plasma stability and release mechanism. The most popular enzymatic clevage sequence is the dipeptide valine-citrulline, combined with a self immolative linker p-aminobenzyl alcohol. Clevage of an amide linked PAB triggers a 1,6-elimination of carbon dioxide and concomitant rlease of the free drug in parent amine form. (Owen, “Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry” AAPS Journal, 17(2), 2015). 

At N-terminal Amino Acid Residue:

Kim (EP 3015116) discloses an antibody-drug conjugate having a drug conjugated to an N-temrinal amino acid residue of a H or L chain of an antibody. Examples of the N-temrinus include not only aino acid residues at the distal end of th N-temrinus but also residues near the N-temrinus. Exampels of the reactive gorup capable of crosslinking with the alpha-amine group include any reactive groups known in the arm which can crosslink with the N-temrinal alpha-amine group of the H or L chain and include isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride. 

At cystein/Lysine/Glutamine residues:

In theory, bioconjugation is possible via most amino acids. Nevertheless, the nucleophilic primary amine of lysine and reactive thiol of cysteine are the most commonly used amino acids inthe bioconjugation of antibodies. Lysine is one of the most commonly used amino acid residues for linking substrates to antibodies, becasue they are usually exposed on the surface of the antibody and thus easily accessible. Alkylation and acylation are the most important reaction with the nucleophilic E-amine. Antibodies can ontain up to 80 lysine residues and as a result, conjugaiton wa lysines inevitably leads to different number of substrates per antibody and antibodies with teh same number of substrates attached at idfferent sites. Furthemore, modifed lysines in proximity to the antibody-bnding site may influence the interaction of the IgG with the corresponding antigen. (In contrast to lysines, the number of cysteine residues within the sequence of IgGs is much lower. Tehre are only 4 interchain disulfide bonds that can be targeted as potential conjugation sites and thus the heterogeneity of immunoconjugates can be dramatically reduced. Moreover, these cysteines form covalent disulfide bonds to stabilize the tertiary sturcture of teh antibody and thus under non-reducign conditions are not reactive. Schibli “Antibody Conjugates: form heterogeneous populations to defined reagents” Antibodies 2015, 4, 197-224). 

Cysteine based conjugation methods rely on a specific reaction between cysteine residues of the antibody and a thiol reactive functional group intalled on the payload. Amide coupling is a major ADC conjugation ethod connecting a payload and solvent accessible lysine residues on the antibody using linkers containing activated carboxylic acid esters. Amide coupling of an amine and an activated carboxylic acid is one of the most reliable high yielding chemical conversions in oranic synthesis. However, there are about 80 lysine residues on a typical antiboy and about 10 reisdues are checmially accessible. Thus, this conjugaiton modality often gives multiple ADC species with variable DARs and conjugation sites. (Tsuchikama, Protein Cell, 2018, 9(1)): 33-46.)

Employing the thiols of interchain cysteine residues in monoconal antibodies as attachment sites for drug molecules if one of the most used conjugation methods. In a human IgG1, there are four interchain disulfide bonds that can be used as potential conjugation sites. The four interchain disulfide bonds can be reduced by tris(2-carboxyethyl) phosphine (TCEP) or dithiothreitol (DT) which results in eight thiol gorups that are available for conjugating drug molecules. Through this method, different drug antibody ratio (DAR conjugates will be obtained when targeting typical DARs of 2-04. Amines of the antibodies can also react with the carboxyls that dervied form the drugs in the effect of the NHS to give antibdoy drug conjugates. Amines of lysines are commonly used for linking drugs to antibodies becasue lysines are usually exposed on the surface of the antibodies and thus easily accessible. Antibodies contain up to 80 lysines and as a resutl, conjugation through lysine residues inevitably leads to heterogenicity such as different number of drugs per antibody and antibodies with the same number of drugs attached at different sites. (Yao “Methods to design and synthesize antibody-drug conjugates (ADCs) International J. of Molecular Sciences, 17, 194, 2016). 

The traditional linking sites in the preparation of protein conjugates are primary amines on lysines. The epsilon amino group in lysine reside can be easily acylated. However, depending on the number and relative accessibility of lysine residues on the protein surface, the random acylation process would generally result in a heterogeneous mixture of conjugates. These challenges have led to the use of thiols for mAb conjugation, as the number of thios is limited and fixed in the different subtypes of IgGs. The commercial antibody products are primarily IgG1s, which possess 4 inter and 12 intra chain disulfide bonds. Drugs can be linked to inter chain disulfides following reduction. Nonethelss, the use of disulfides for conjugation may still yield a hterogeous populaiton of conjugated products that contains positional isomers. Goswami (Antibodies 2031, 2, 452-500)

Cytotoxic drugs are generally conjugated to antibodies either thorugh lysine side-chain amines or through cysteine sulfydryl groups activated by reducing interachain disulfide bonds. Both of these procedures yield heterogenous products containing a mixture of species with different molar ratios of drug to antibody, linked at different sites, each with distinct in vivo pharmaokinetic, efficacy and safety profiles. To limit the potential liabilities associated with such conjugation methods, Junutula engineered reactive cysteine residues at specific sites in anitobodies, termed THIOMABs”, to allow drugs to be conjugated with defined stoichiometry without disruption of interchain disulfide bonds. For conjugation to covalently attach thiol reactive probes to engineered cysteines, first, the cysteine and glutathione adducts were removed from the THIOMABs by partial reduction followed by diafiltration. This partial reduciton also disrupts interchain disulfide bonds but not intrachain disulfide bonds. The interhain disulfide bonds were allowed to reform by air oxidation or by accelerated oxdiation using CuSO4 of dehydro-ascorbic acid. After this treamtnet, THIOMAB variants were then conjugated with biotin-PPO-maleimide. (Junutul “Site-specific conjugaiton of a cytotoxic drug to an antibody improved the therpaeutic index” Nature biotechnology, 24(8), 2008).

Hu (WO 2015/1091883) discloses an Fc cotnaining polypetpide such as an antibody which includes an N-glycosylated Fc region that includes an acceptor gluamine residue flanked by an N-glycosylation site and where the conjugate moiety is conjugated to the Fc containing plypeptide via the acceptor glutamine residue. The term “acceptor glutamine residue” refers to an amino acid that under suitable conditions is recognized by a transglutaminase (TGase) and can be crosslinked to a conjugate moeity that includes a donor amino group by a TGase through a reaction between the glutamine and donor amino groups (such as lysine or a structurally related primary amine such as amino pentyl group). The conjugation between the conjugation moeity and the acceptor glutamine residue is carried out by conjugating the amine donor group of the conjugation moeity or the small molecule handle to the acceptor glutamine residue. Thus, any conjugate moeity containing an amine donor group can be directly conjugated to the Fc bontianing polypetpide. Any conjugate moeity not containing an amine donor group can be indirectly conjugated to the Fc containing polypeptide via a small molecule handle which cotnains an amine donor group. For example, the conjugate moeity can include an amine donor group (e.g., primary amine-NH2), an optional linker, and an active moeity (e.g., a small molecule) The conjugate moeity can also be a polypeptide or a biocompatible polymer containing a reactive Lys. The amine donor group such as a primary amine (-NH2) provides a substrate for transglutaminase to allow conjugation of the agent moetiy ot the Fc contiaining polyeptide via the acceptor glutamine. Accordingly, the linkage between the donor glutamine and the amine donor group can be of –CH2-CH2–CO–NH–. In some embodiments, the Fc containing polypetpide and the conjugate moeity are linked throguh a cleavable linker. In some embodiments, the conjugate moeity includes an active moeity that is a cytotoxic agent. In some embodimetns, the N-glycosylation site and the acceptor glutamine resiude are 5 or less amino acid residues apart.

Shen (Nature biotechnology, 30(2), 2012) discloses generation of antibodies with cystein residues engineered into the IgG H chain that provide reactive thiols for conjugation to the auristatin and maytansine classes of cytotoxic drugs. 

Thiol maleimide linkage has been widely used in the synthesis of ADCs because of its high slectivity, rapid reaciton kinetics and compatibility with aqueous reaciton conditions. For example, Bentuximab vedotin is prepared by reducing the endogenous antibody cystines, followed by conjugation of cysteine thiols with a maleimide-funcitonalized monomethyl auristatin D derviative. (pone, “understanding how the stbiliyt of the thiol-maleimide linkage impacts the pharmacokinetics of Lysine-linked antibody-maytansinoid conjugates). Biconjugate Chemistry, 2016). 

Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics) are produced. by conjugation to surface exposed lysines or partial disulfide reduction and conjugaiton to free cysteines, respectively. These stochastic modies of conjugaiton lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. (Zimmerman, “Production of stie-specific antiboy-drug conjugates using optimized non-natural amino acids in a cell free expresison system). 2014). 

Matsuda (US 2021/0139541) discloses compounds having an affinity substance to an antibody and a bioorthogonal functional group of the formula A-L-E-B where B is a bioothogonal functional gruop, E is a divalent group that includes an electrophilic group coupled with a leaving group and having ability to reactive with a nucleophilic group in the antibody, L is a divalent group that incldues a leaving rup and A is an affinity substance to the antibody. 

–Ezyme-directed Modification:

Chilkoti “A noncanonical function of sortase enables site-specific conjugation of small molecules to Lysine residues in proteins” Angew, Chem. Int. Ed. 2015, 54, 441-445) disclsoes that the enzyem sortase A can be used to conjugate small molecules to a mAb with an ezquisite level of control over the site of conjugation. Sortase A recognizes the primary sequence LPXTG (where X is any amino acid) in a protein and cleaves the peptide bond between threonine and glycine, forming a stable intermedaite that joins the catalytic thiol in Sortase A to teh carboxyl group of threonine in a thioster bond. Chilkoti cloned the 4D5 mAb against human epidermal growth factor receptor 2 (Her2) and genetically modifed it to contain a pilin domain at the carboxy termini of its H chain. The recombinant antibody was incubated with SortageA-ELP and biotin-LPETGRAGG peptide overnight. Only the anti-Her2 H chain modifed to contain the pilin domain was biotinylated by SortageA-ELP. 

–Affinity Guided Conjugation:

Affinity guided conjugation strategies invovle the incorporation of a scaffold that enables non-covalent interactions to occur between the affinity reagent and the target protein. This, in turn, increased local concetnraiton ofa.reactive moeity to a particular site, allowing for site-specific modificaitons to take place. Chilkoti “A noncanonical function of sortase enables site-specific conjugation of small molecules to Lysine residues in proteins” Angew, Chem. Int. Ed. 2015, 54, 441-445)

Small domains that bind with high affinity to a conserved sequence in teh Fc domain of antibodies are used to form non-covalent antibody conjugates. This approach does not require any modificaiton of the antibody. Among numerous Fc binding domains, teh ZZ domain –a dimer of the modified immunoglobulin binding site of protein A is the most widely used. Its high affintiy interaciton with the Fc part of antibodies has been exploited to by genetically fusing the ZZ domain to Pseudomonas exotoxin A. Incubaton with a antibody yielded hihgly toxic non-coavlently coupled ADCs. Schibli “Antibody Conjugates: form heterogeneous populations to defined reagents” Antibodies 2015, 4, 197-224).

Ito (US 2018/0141976 and EP 3299383) dicloses an IgG binding peptide having an amino acid sequence of 13-17 amino acid residues having the formula (X1-3)-C-(X2)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(X1-3). Xaa1 is a protein constituting amino acid such as a lysine residue, a cysteine residue, an aspartic acid or a glutamic acid residue or a non-protein constituting amino acid such as diaminopropionic acid or 2-aminosuberic acid. It is preferred that Xaa1 is modified with a cross-linking agent. The cross-linking agent is a chemical substance for linking the IgG binding peptide to IgG Fc via a covalent bonds. This can be a compound having at least two sites capable of binding to the desired amino acids (such as Lysine, cysteme). Examples include cross-linking aagents containing two or more succinimidyl grups such as DSG and DSS, cross-linking agents containing two or more imidic acid moieties such as DMA. It is also preferred that the peptide has no or little the same residue as Xaa1 (e.g., has only one or two same residues as Xaa1) in the sequence. Two cycteins in the peptide can form a disulfide bond to form a cyclic peptide. Alternatively, sulfide groups in the two cysteine residues on the other sides of the peptide may be linked via linker. The peptide can then be mixed with IgG causing a cross-linking reaction of the petide with the cross-linking agent with the IgG. This eliminates the need of altering the sequence of the antibody molecule. The IgG binding petpide can be modified with additional functional substances such as a labelling agent or an additional drug. The cross linking to IgG can occur site specifically between the amino acid residues Xaa1 of the IgG bidning peptide and Lys248 or Lys246. 

Yamada (US Patent Applicaiton No: 16/663,791, published as US 2020/0190165) discloses an antibody which includes lysine residue(s) in a target region and 5 or more lysine residues in a non-target region and at least one functional substance which is a drug, a label or stabilizer which binds to the lysine residue(s) in the target reigon with 30% or more regioselectivity through a linker which does not include a peptide portion. The antibody is prepared by preparing a compound haivng the formula A-L-B-R where A is an affinity substance to a soluble protein, L is a cleavable linker which is a divalent group comprising a cleavable porition, B is a divalent group that includes a bioorthogonal functional group or a divalent group with no bioorthogonal functional group and R is a reactive group to a soluble protein such as an antibody. The reactive group can be specific to a side chain of a lysine residue. The bioorthogonal functional groups can bind to the soluble protein via a side chain of a lysine, tyrosine or tryptophan residue. First a soluble protein (T) such as an antibody assocaites with the compound through an affinity substance (A) to the soluble protein. Next, the compound reacts with a side chain of a specific amino acid residue (e.g., a side chain of a lysine resiude) in a target region present near an association site of the affinity substance and the coluble protein through a reactive gorup (R) (e.g., an activated ester) to form a conjugate between the compound and the soluble protein. The cleavable portion enables relase of the affinity substance (polypeptide) to an antibody. 

Besides pH, buffer components also play a role in stabilizing mAbs. Sodium phosphate and histidine are the most commonly used buffer components in commerical formulations. While sodium phosphate is more common for low concentraiton mAb formulations, histidine is the primary buffer of choice for higher mAb concentration. Goswami (Antibodies 2031, 2, 452-500)

Carpenter (WO2008/039761) discloses stable liquid antibody formulations of antibodies comprising non-zwitterionic buffers such as phosphate (e.g., Na3PO4), tris, citrate, succiante and acetate buffers. 

Ionic strenght, pH and Temperature Considerations: 

The first step in formulation development is pH optimziation. The most common approach is to identify the pI of the mAb form the primary sequence using common available software such as ExPASy or Sednterp, followed by formulation of mAb at pH away form the pI. Various pathways of chemical degrdation are pH sensitive. For example, deamidation and isomerization are favored at neutral and basic pH. Thus it is preferably to formulate biopharmaceuticals at slightly acidic pH. In fact, out of 28 commercially approved mAb type biopharmaceuticals, 20 are formulated at pH less than or equal to 6.5. Goswami (“Developments and challenges for mAb-based therapeutics” Antibodies 2013, 2, 452-500)

US 4,3596,608 and 4,499,073 disclose that in order to obtain an immunogloublin composition which is substantially free of an aggregate of immunoglobulin and has a monomer content of immune serum globulin exceeding about 90%, a solution of the immune serum globulin is adjusted to have an ionic strenght less than about 0.001 and a pH of 3.5 to 5.0.

JP-A-7-238036 discloses that for the improvement of stability, the use of acid treatment or storgage at room temperature.

disclose that conformational stability of IgG preparations decreases with decreasing pH, while the resistance against aggregation improves. The optimum pH range for storage was 5-6, as a compromise between conformational stability and the tendency for oligomerization. Biologicals 34 (2006) pp. 5-14). 

Importance of isoelectric point: (see also engineering Abs to alter pI)

One important characteristic of mAbs is their isoelectrin point (pI) which essentailly is the pH at whcih teh antibody has no net electrical charge and its value depedns on the charged amino acids the antibody contains. If the pH of the surrounding environment is below the antibody’s pI, then the molecule carries a net postive charge wehreas the antibody will carry a net negative charge when the pH is above the pI. It is generally observed that increases in net positive charge of antibodies result in increased blood clearnace and increased tissue retention with shorter half-life, whereas antibodies with lower pI generally have decreased tissue uptake and longer half-life, although observations can be conflicting regarding correlation between mAb learance and pI. Even subtle manipulation such as molecular surface remodeling to disrupt positive path regions can influence pK properties. (Schneider “Important of isoelectric point (pI) of antibodies, 2017. 

There is interest in identifying antibody candiddates with highly repulsive seelf-interactions in stadnard formualtions (e.g., low ionic strengh buffers at pH 5-6) fo high solubility and low visosity. Likewise, there is interest in identifying antibody candidates with low levels of non-specific interactions in physiological solution condition (PBS, pH 7.4) to promote favorable phamacokinetic properteis. (Gupta “Antibodies with weakly basic isoelectric points minimize trade-offs between formulation and physiological colloidal proeprties” Mol. Pharmaceutics 2022, 19, 775-787). 

Gupta “Antibodies with weakly basic isoelectric points minimize trade-offs between formulation and physiological colloidal proeprties” Mol. Pharmaceutics 2022, 19, 775-787) discloses that IgG1 wieth weakly basic isoelectric points between 8 and 8.5 and variable fragments (Fvs) isoelectirc points between 7.5 and 9 typically display the best comibnations of strong repulsive self-interactions and weak non-specific interactions. 

An inherent protein property of interest is its isoelectric point (IEP). The IEP is defined as the pH at which net charge becoems azero. It is a rule of thumb to define the formulation’s pH not too close to IEP as this increases the risk of preciptiation. The IEP of prtoeins can be theorecctically derived from amino acid sequence or determined experimentally using different methods including isoelectric focusing (IEF) of IEF capillary electrophoresis (iCE). (Stefan fischer “Assessmetn of net charge and prtoien-protein interactions of different monoclaonl antibodies” J. Pharma Sciences, 100(7) 2011).

Addition of Amino Acids:

Most of the liquid IgG which is formulated with amino acids on the market must matinain an acid pH to avoid aggregation, preferably between a pH of 4-50 at at a temperature of 2-8C if they are stabilised with 0.2M or 0.25 M glycine such as Gamunex®, Kiovig® or Gammard® lquiud (both from Bater) or up to 25C if stablized with 0.25M proline such as Privigen® (CSL Behring, Germany).  However, an excessively acid pH over an extended period of time favours fragmentation of IgG. Ristol Debart (US13/838424). 

Arginine: Liu (US 2007/0053900) discloses that arginine, specifically arginine-HCl is particularly suited for highly concentrated liquid protein or antibody formulations.

Glycine and/or histidine/ serine: 

Kakuta (US 2003/01901316) discloses a stabilized preparation containing an antibody in a glycine and/or a histidine buffer.

Yang (WO2004/001007) discloses a composition of antibodies in aqueous solution of histidine or acetate buffer at a concentration in the range of 2-48 mM. 

Zhou (US2014/0056888) teaches stable aqueous solutions having a high concentraiton of anti-C5 having a buffering agent which is an amino acid such as histidine, erine and glycine. 

Histidine and Glutamate (His + Glu):

Ignatius (J. Pharma Sciences, 108 (2019) 11391147 (2019) discloses systematic buffer screening for an aggregation prone bispecific antibody and finding that equimolar combiantion of histidine and glutamate at pH 5 bufer showed enhanced colloidal stability as measured by dyanmic light scatering interaction parameter (Kd). This implies a role of net protein-protein interaction in mediating aggregation propensity of the protein. The His + Glu interaction with the protien is predominantly electrostatic in nature as observed by the switch in the net protein-protein interactions from repulsive to attractive interactions in the presence of 20 mM NaCl. 

Histidine and NaCL

The presence of 50 mM sodium chloride in 10 mM histidine pH 5.6 was found to increase diffusion interaction parameter (KD) or reduce self-interaction, improve relative solubility and reduce turbidity (OD350) of an anti-LAG3 antibody. The stability of anti-LAG3 antibody in 10 mM histidine pH5.6 was investigated in the presence of 40 mM L-arginine hydrochloride using diffusion interaction paraemter (KD), tubidity and relative solubility. The self-interaction and turbidity was found to be dramatically reduced. 

 Methionine/Tryptophan: 

Junyan (US13/536584) discloses a method of stabilizing and preventing oxidation of aromatic amino acid residues within a susceptible antibody by adding methionine in combination with one or more vitamins.

Methionine is an antioxidant that prevetns antibody oxidation durin gprocessing and storage (Ma US 8,613,919)

Ramachandra (US2011/0097340A1) teaches anti-VEGF antiboides which are formulated with antioxidants such as methionine.

Sato (EP1260230A1) teaches stabilization of antiboides and proteins such as GCSF by adding methionine and tryptophan. The composition can also include surfactants and mannitol.

Sloey (US2010/0297117A1) discloses stable formulations of antibodies having one or more antioxidants including methionine, aromatic amino acids such as tryptopyhan and vitamins such as water soluble vitamin B6. Sloey further teaches that the formulations may optionally include surfactants and mannitol.

Warne (US2006/0193850) teaches stabilization of antibody with methionine as an antioxidant in an amount sufficient to inhibit the formation of undesired by-products.

Phenylalanine, proline, leucine and isoleucine: Bruegger (CN1157572 (abstract) disclsoes liquid preparations of IgG for intravenous infusion which are stabilixed against dimer formation by the addition of one or more amphiphilic stabizers. Preferred amphiphilic stabilizers are nicoteinic acid derivatives, particularly nicotinamide and naturally occuring alpha amino acids having a lipophilic side chain such as phenylalamine, proline, leucine and isoleucine.

IgPro10 is a liquid intravenous immunoglobulin (IVIG) product that is formulated with 250 mM L-proline at pH 4.8 (Vox Sanguinis (2009) 96, 219-225. 

Addition of Carbohydrate/Polyols or Poly-alcohols

Formulation of IgG with polyols or poly-alchohols, for example, with maltose and sorbitol prevents aggregation. IgG solutions that are stable up to 25C (with 10% maltose, trade name Octagam®) and up to 30C with 5% sorbitol (trade name Flebogamma®) have been formulated in a slightly acid pH range between 5-6. However, the presence of some sugars in IgG formulations has been questioned. Some cases of kidney failure have been associated with preparations containing saccharose.  Ristol Debart (US13/838424).

Lam (US6,171,586) describes a stable aqueous pharmaceutical formulation having a therapeutically effective amount of an antibody not subjected to prior lyophilization, a buffer maintaining the pH in the range from about 4.5-6, a surfactant and a polyol. 

Addition of Sorbital and Mannitol: 

JP-A-63-192724 discloses a liquid immunoglobulin composition for intravenous injection having low conductivity and pH of 5.5 ±0.2 and containing sorbital as a stabilizer.

Barrera (60 Ann. Rheum. Dis. 660-69 (2001) discloses s single does of antibody to study short term effects in rheumatoid arthritis patients, using a preparation of 25 mg/ml mAb in 1.2% mannitol, 0.12% citric acid, 0.02% sodium citrate in an intravenous infusion. 

Hirao (EP0278422B1) teaches an intravenously injectable solution of gamma-globulin in solution containing 5 to less than 10% by wweight per volume of sorbitol as a stabilizer and having an electrical conductivity of not higher than 1 mmho and pH of 5.5.

Zhou (US2014/0056888) teaches highly concentration antibody formulations having at least one carbohydrate excipient such as sorbitol and mannitol. 

 Addition of Polymers:

Polyelectrolytes:

Gibson (WO95/10605) discloses stabilisation of proteins such as antibodies and enzymes which includes a polyelectrolytes. The polyelectrolyte can be cationic, anionic or amphoteric. 

–Anionic Polyelectrolytes:

Gibson (WO95/10605) disclsoes stablisation of rptoeins such as antibodies with anionic polyelectrolytes which are preferably a polymer with anionic groups distributed along the molecular chain. The anionic groups, which may include carboxylate, sulphonate, sulphate and other negatively charged ionisable groupings may be disposed upon groups pendant from the chain or bonded directly to teh polymer backbone. 

–cationic polyeletrolytes:

Gibson (WO95/10605) disclsoes stabilisation of proteins such as antibodies which include cationic electrolytes. The cationic groups, which are preferably quaternary ammonium derived functions may be disposed in side groups pendant form the chain or may be incorproated in it.  Examples of caitonic polyelectrolytes include coplymers of vinyl pyrollidone and quaternary methyl methacrylate. 

—–Polyamino compounds (e.g., spermine and spermidine):

Spermidine is an aliphatic polyamine. Spermidine synthase catalyzes its formation from putrescine. It is a precursor to other polyamine, such as spermine and its structural isomer thermospermine (Wikipedia). 

Bowen (US 2013/0058958) discloses using certain charged amiono acids for reducing the viscocity of aqueous protein contianing formulations. In oueous formulation is spermidine or spermine. ne embodiment the protein is an antibody and the the compound that is capable of reducign the viscosity of the aq

Gelder (WO 2006/094974) disclsoes a pharmaceutical composition that incldues a stabiliser composition with a biological molecule such as an antibody. In a preferred embodiemtn, the stabiliser composition includes at least one polyamine (compound with two or more amino groups) such as spermidine and psermine. The composition can also include a sugar such as glucose, lactose, sucrose, maltose osorbitol or mannitol and an amino acid such as glutamate (gluamic acid) or glycine. The composition is particularly useful for stablising vaccines. 

Gibson (WO95/10605) discloses stabilisation of proteins such as antibodies which includes low molecular weight polyamino compounds such as spermine and spermidine. 

Polyethyleneglycol (PEG): JP-W-59-501546 discloses ultrafiltration treatment of an immunoglobulin prepration at pH 5 to 5.6 in the presence of 0.05 to 2 w/v % polyethyleneglycol (PEG).

US 5,132,406 discloses a method for the production of immunoglobulin preprations for intravenous injection, which comprises a combination of a heat treatment step, a supernantant fraction recvoering step by a fractionation treatment with 4 to 10% PEG and a preciptaition fraction recovering step by a 10 to 15% PEG fractionation treatment.

Hirao (US 6,159,471) discloses an immunoglobulin prepration having good storeage stability in the form of a solution by fractionating with an aqueous solution with 4 to 10 w/v % of PEG haivng a MW of from 1k to 10k at a pH of from 4.5 to 6.5 at an ionic strenght of from 0.0001 to 0.1 M and a temperature of from 0 to 4 degrees C.

Addition of Surfactants

Surfactants are also included in typical mAb formualtions. Surfactants are surface active agents that are amphipathic in nautre (polar head and hydrophobic tail). Surfactants tend to accumulate at various interfaces, resulting in reduced interfacial tension. Goswami (Antibodies 2031, 2, 452-500)

Addition of Non-Ionic surfactants

WO95-3826 discloses the immunogloublin preparation comprising 0.1 g/L or less of non-ionic surfactant as stabilizer for maintaining solution state, and being substantially free of albumin.

–polysorbates: 

Lam (US6,171,586) discloses a stable aqueous formulation of an antibody with a buffer maintaining the pH in the range of about 4.5-6 and a surfactant such as polysorbants (polysorbant 20, 80, etc) which reduce aggregation.

Zhou (US2014/0056888) teaches hingly concnetrated antibody formulations having a surfactant such as polysorbate 20 or polysorbate 80. 

There are various methods available for assessing the stability of protein formulations, incluidng antibody formulations, based on the physical and chemical structures of the proteins as well as on their biological activities. Methods include charge-transfer adsorption, thermal analysis, fluorescence spectroscopy, circular dichroism (CD), NMR, and HPSEX, tangential flow filtration (TFF), static light scattering (SLS), fourier transform infrared spectroscopy (FTIR), urea induced protein unfolindg techniques, intrinsic tryptophan fluorescence, differential scanning calorimety and I-anilino-8-naphthalenesulfonic acid (ANS) prtoein binding techniques. (Carpenter, WO 2008/039761). 

Forced degradation assays of mAbs are frequently performed to support formulation development of mAbs. The commonly used forced degradation conditions include high temperature, freez-thaw, agitation, high pH, low pH, light exposure, oxidation and glycation. SEC is the method of choice to detect aggregation and to a lesser degree, fragmentaiton. Charge-based methods such as CEX or isoelectirc focusing electrophoresis are very sensitive to the detection of overall changes to the molecule, mianly due to deamidation but from other modificaiton as well. Liquid chromatography-mass spectrometry (LC-MS) analysis of the intact or reduced MW is also very useful for detection of modificaitons such as oxidation and glycation. (Liu, “Forced degradation of recombinant monoclonal antibodies: a practical guide” MABS 2017, 9(8) 1217-1230). 

Monitoring Protein Aggregates

reducing capillary gel electrophoresis (rCGE) and high performance size exclusion chromatography (HPSEC) are the most common and simplest methods to assess the formation of protein aggregates, protein degradation and protein fragmentation. For example, the percent area of the peaks represent the non-degraded antibody or non-degraded antibody fragments. (Carpenter, WO 2008/039761; Bishop (US 2010/0209434))

Charge Variants

Ion Exchange High Performance Liquid Chromatography (IEX-HPLC): charge variants can be separated by IEX-HPLC. The relative amount of a specific protein impurity, expressed as area %, can be calculated by dividing the peak area corresponding to the impurity by the total integrated area. (Brige (US 2014/0178383)

–Cation-exchagne high performance liquid chromatography (cIEX-HPLC): is a technique that relies on charge-charge interactions between a protein and the charges immobilized on the resin. It takes advantgage of the positvely charged ions of a protein that bind to the engatively charged resin. A common structural modificaiton is deamidation of asparagine (Asn) residues and cEX-HPLC permits the separation of deamidated and deamidation intermediates. (Hays US 2008/0113914)

Water Soluble Synthetic Polymers

PEG

Introduction: 

Water soluble synthetic polymers have been used for protein stabilizing agents.s Poly-(ethylene glycol) (PEG) is one such polyer that has been extensively studies both as a protein stabilizer and a protein precipitant. The polymer, at high concentraiton of more than 10% can lead to precipitation but at concentrations less than 1% binds to proteins upon denaturation and can stabilize them against aggregation. Gombotz (Pharm Res. 1994, 11(5), 624-32)

Polyethylene glycol (PEG) conjugation (see also “polymers” under “biochemistry”) is used to alter the pharmacokinetic profiles of a variety of drugs and thus improve their therapeutic potential. PEG conjugation increases retention of drugs in the circulation by protecting against enzymatic digestion, slowing filtration by the kidneys and reducing the generation of neutralizing antibodies (Fishburn, J. Pharma. Sciences, 2008, p. 1). PEG has mainly be used to reduce the immunogenicity and increase the circulating half-lives of antibodies (Chapman, Advanced Drug Delivery Reviews 54 (2002) 531-545).

PEG is a polymer having the properties of solubility in water and in many organic solvents, lacks toxicity and immunogenicity. One of of PEG strategies is to covalently attached the polymer to insoluble molecules to make the resulting PEG molecule “conjugate” soluble. For example, it has been shown that the water insoluble drug paclitaxel when coupled to PEG becomes water soluble. (Roberts, US 6,436,386). The polymer modificaiton tends to reduce needed dosage and dosing frequency and development  of drug directed immune responses, thus improving both short and longer term patient tolerance to treatment. In some cases (e.g., PEG insulin, it may also allow for the drug to be taken orally, parenterally or other routes than by injection. The result is PEGylated drug formualtions which have greater chance of FDA approval, patient tolerance, ease of application, wider patient population (e.g., sales base) and longer safety and efficacy.  (Van Alstine (WO2011/035282). 

Poly (vinylpyrrolidone) (PVP): 

Gombotz (Pharm Res. 1994, 11(5), 624-32) discloses using size exclusion high performance liquid chromatogrpahy (HPLC), differential scanning calorimetry (DSC) and ELISA to evaluation of different concentraitons of PVP as a stabilizing agent for an antibody against heat induced aggregation. Higher concentration of PVP increased aggregation and eventually lead to precipitation but low concentraiton stabilized the antibody against heat induced aggregation. 

Examples:

There are now several “PEGylated” proteins which are FDA approved and have yearly sales in excess of one billion dollars. Examples include “EGINTRON (PEGylated INTRON) from Schering, PEGASYS (PEGylated ROFERON) from Roche and NEULASTA (PEGylated Nuepogen) from Amgen. 

Other Hydrophilic polymers

In addition to PEG other hydrophilic polymers such as those containing mixtures of propylene glycol and ethylene glycol units may also be useful for modificaiton of biopharmaceuticals. So too these netural hydorphilic polymers appear to mimic hydrophilic carbohydroate polymers which might occur as a result of glycosylation and also increase the serum half life of biopharmaceuticals. There are also polymer conjugates of ethylene glycol units and carbohydrate units which are like PEG useful as nonimmunogenic polymers in formulations. One well known example is ethylhydroxyethylcellulose (EHEC). Another is use of dextran (polyglucose) polymer. (Van Alstine (WO2011/035282)

Block Polymers: 

Antibody stabilization by addition of block polymers composed of polyoxypropylene and polyoxyethylene in combination with phospholipids is described in EP-A 0318081.

Antibody Fragment PEGylation

Fab’ fragment PEGylation:

Antigen-binding fragmetns (Fab’) of antibodies can be site specifically PEGylated at thiols using cystein-reactive PEG-maleimide conjugates. For therapeutic Fab–PEG, conjugation with 40 kDa of PEG at a single hinge cystein has been found to confer appropriate pharmacokinetic properties to enable infrequent dosing. (Humphreys, Protein Engineering Design & Selection, 20, 5, pp. 227-234, 2007). 

See aslo antibody variants in Antibody purification

Unpredictability in Antibody Formulations/stabilization

mAbs generally have reasonable stability, and about half of all mAb drug products are in liquid dosage form. Goswami (Antibodies 2031, 2, 452-500).

If one examines the amino acid sequences for IgG1 based antibody drugs, only a small segment of these proteins are dramatically different one one another, being the Fv segments that involved in antigen binding. One might assume that by finding a stable formulation for one of these antibody drugs, such a formulation would be good for most if not all, similar antibodies. Instead, each antibody seems to have a unique persoanlity related to its requirements for stability; a phenomenon derived form the fact that the small differences between these antibodies are fucoused on surface epxosed amino acid differences that stipulate antigen specificity. Thus, the interfacial surface of each antibody is unique and requires specific formulation components to provide maximal stability and retention of activity. (Dougherty, Advanced Drug Delivery Reviews 58 (2006) 686-706). 

Despite acknowledging the similarity in structures, Wang repeatedly states that the differences amoung antibody sequences affect the stability of antibody pharmaceuticals. One formulation excipient stabilizing a specific antibody may not be suitable for another because of the differences in their sequence. (Wang, “Antibody Structure, Instability, and Formulation, 96 U. Pharm. Sciences  1-26 (2007) (Ex. 2007); See also IPR2016-01018, Patent 9,114,166B2 which discusses Wang as evidentially support that one of ordinary skill in the art would hnot have had a reasonable expectation of success in formulating a stable, liquid, high cconcentration D2E7 formulation as required by the claims. ). 

Liquid Formulations

Developing conditions to keep proteins stable in a liquid form for a pharmaceutically relevant storage time (e.g., two years) is not a simple task. With many proteins, it is not possible, especially considering the time constraints for product development, to develop sufficiently stable aqueous formulations. (Rational design of stable protein formulations, 13 Pharmaceutical Biotechnology (John F. Carpenter & Mark C. Manning eds., 2002) See as IPR2106-01018, Patent 9,114,166B2 which discusses this book). 

The development of stable liquid antibody formulations, especially those at a concentration high enough to be suitable for [subcutaneous] administration, was far from routine (Wang, Instability, Stabilization, and Formulation of Liquid Protein Pharmaceuticals, 185 Int’l J. Pharmaceutics 129-188 (1999). 

Types of changes which can occur to Antibodies

Chemical degrdation of antibodies may occur in a number of different ways. The type of degradation affects the primary sequence and may also lead to significant changes in the higher order structure. Examples of chemical degradation include deamidation, oxidation, isomerization, clipping/fragmentation and cross-linking. Degradation of the molecule may interfere with the intended biological activity, as the site of degradation may involve a domain that is critical for biological function. There are cases in which the formation of aggregates was followed by loss of biological activity, as for example antiviral activity of interferon-beta-1b. Another major impact of protein aggregation is immunogenicity, which may result from the multipolicity of the epitope and/or change in conformation. Anitobides formed can be nuetralizing in nature and thus may simply result in the loss of efficacy of the adminsitered therapeutic. if the level of nuetralizing antibody is very high, it may result in the neutralizaiton of the essential endogenous protein, for example, the nuetralization of endogenous erythropoietin by the antibody, formed against recombinant human erythropoietin has been reported to cause red cell aplasia Goswami (Antibodies 2031, 2, 452-500)

According to the International Conference on Harmonization (ICH) guidance document, drug substance heterogeneity defines its quality, and the degree and profile should be monitored and characterized to ensure lot to lot consistency. Microheterogeneity of mAbs thus is a concern for production, expecially for downstream processing. Characterization of the product is necessary for determining variants.  (Burg, WO2011/009623). 

A number of authors have shown that charge variants of therapeutic proteins can have vastly different bioactivity (Pabst, Biotechnol. Prog 2008, 24, 1096-1106). 

Oxidation: methionine and cysteine residues are frequently a site of oxidation in protein drugs and this is also the case for antibody drugs. Specific methionine reisudes within the Fc domain may be prone to oxidation, resulting in the production of methionine sulfoxide. While cysteines are present in teh Fc framework as disulfide pairs, unpaired cysteines in the variable region may also be sites of oxidaiton. Besides methionine and cysteine residues, oxidation of histidine, tyrosine, tryptophan and phenylalamine reisudes can also occur. (Dougherty, Advanced Drug Delivery Reviews 58 (2006) 686-706). 

 Modifications that form acidic species

Acidic species are antibody variants that elute earlier than the main species of the antibody during CEX or later than the main peak during AEX analysis. The main causes for the formation of acidc spces include sialic acid, deamidation, glycation (Du, “Chromatographic analysis of the acidc and basic species of recombinant monoclonal antibodies” Merck Research Laboratories (2012). 

–Deamidation of side chains of asparagines aand glutamine residues:  

Deamidation is probably the most common type of chemical degradation encountered in mAb based biotherapeutics. Goswami (Antibodies 2031, 2, 452-500). Glutamine and asparagine residues show a propensity for deamidation. Initial detection of deamidation in antibody preparations is typically dientified by differences in charge distribution of content using methods such as isoelectric focusing (IEF) of high performance cation exchange chromatography (Dougherty, Advanced Drug Delivery Reviews 58 (2006) 686-706). 

Deamidation in proteins is often responsible for charge heterogeneity of recombinant proteins. The uncharged side chains of these amino acids are modified to an isoglutamate and iso-asparatate residue to a glutamate and aspartate residue. Thus, an additional charge is introduced to the protein per modification.  For recombinant monoclonal antibodies, deamidation can occur at any stage from inside the cells, after secretion, during purification, during storage and under different conditions of stress.  Deamidated and other acidic variants of the her2 antiboides, for example, were found to exist in seven different variants and the variants could be separated by cation exchange chromatography using a liner increase in ionic strengh for elution.  (Burg, WO2011/009623). 

Glycosylation pattern changes: can also occur in the course of cell culture (Burg, WO2011/009623).

Factors which Influence Antibody Stability:

Nonspecific protein-protein interactions have been driven by the need for developing stable liquid formulations of antibodies. Antibody self-interactions are linked to problems such as protein aggregation and high viscosities. These problems are often exagerated at high protein concetnrations, which are often needed in liquid formulations to meet patient dose requirements. Problems can be minimized by manipulating the colution condtiions, such as choice of buffers, pH, ionic strength, and the inclusion of other small molecule additives sucha s sugards, amino acids and other excipients. (Curtis “The role of electrostatics in protein-protein interactions of a monoclonal antibody” 2014). 

Isoelectric point: 

An inherent protein proeprty of itneret is its isoelectric point (IEP). The IEP is defined as the pH at which net charge becoems azero. It is a rule of thumb to define the formulation’s pH not too close to IEP as this increases the risk of preciptiation. The IEP of prtoeins can be theorecctically derived from amino acid sequence or determined experimentally using different methods including isoelectric focusing (IEF) of IEF capillary electrophoresis (iCE). (Stefan fischer “Assessmetn of net charge and prtoien-protein interactions of different monoclaonl antibodies” J. Pharma Sciences, 100(7) 2011). 

Techniques used to Increase Antibody Stability

A number of additive have been identified that can reduce the rate of protein aggregation including urea, guanidinium chloride, amino acids (in particular glycine and arginine), various sugars, poly alcohols, polymers (including polyethylene glycol and dextrans), surfactants and even antibodies themselves.  (Dougherty, Advanced Drug Delivery Reviews 58 (2006) 686-706). 

(1) Addition of Amino Acids:  See Buffer conditions

(2) Lyophilization: 

Because immunoglobulin is unstable in the form of a solution, it has been formulated not as a liquid preparation but as a drug preparation. However, the dry prepration is accompanied by the problem that it cannot be administered easily because of the necessity of dissolving it in distilled water for injection.

(3) Addition of Carbohydrate/Polyols or Poly-alcohols:  See buffer Conditions

The modification of the carbohydrate chains and thus the glycosylation of proteins is done in order to improve the erum half life of recombinantly expressed proteins such as antibodies. Sialic acids are the most prevalent terminal monosaccharides on the surface of eukrayotic cells and it is generally believed that the more a glycoportein is sialyated the longer is its serum half life during ciruculation. Goletz (US2010/0028947). 

(4) Addition of Sorbital and Mannitol:  See buffer conditions

(7) Addition of Polymers:  See buffer conditions

(8) Non-Ionic surfactants: See buffer conditions

Formulation of IVIG

Deamidation and isomerization are favored at neutral and basic pH. Thus it is preferable to formulate biopharmaceuticals at slightly acidic pH. In fact, out of 28 commerically approved mAbs, 20 are formulated at pH less than or equal to 6.5. (Goswami, Antibodies, 2013, 2, 452-500, 2013)

IVIG has enhanced stability in the liquid state at pH 4.25. There is a trend for modern IVIG preparation to be formulated as liquid at high protein concentrations (typically 100 mg/ml) within a low pH range (pH 4.5-5.5) in the presence of stabilizers like polyols (sorbitol), sugars (maltose, glocose) or amino acid (glycine, proline, isoleucine) and without sodium chloride addition. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28.