Companies working on protein stabilization: Ambient
See also Immunoglobulin stabilization and Buffer conditions
Importance and Difficulty of Drug Delivery and Formulation
The success of most peptide and protein drugs is dependent upon the delivery of the biologically active form to the stie of action. In the design and developmnet of formulations to acheive this goal, the formulation scientist must consider the clinical indicaiton, pharmacokinetics, toxicity and physicochemical stability of the drug. The development of a stable formulation is a necessary step for each new protein or peptide therapeutic. The degradation pathways and their impact on stability should be systematically analyzed and competing degradation rates must be balanced to arrive at the most stable formulation posible. The design and production of protein and peptide drug formulations is not well developed and many of the mechanisms for stabilization and delivery of these drugs have not been determined. The route and fequency of administration and the bioactivity or portency of the drug in humans are critical issues that are often not addressed in the pre-IND animal studies. (Cleland and Langer, “Formulation and Devliery of Proteins and Peptides,” American Chemical Society, 6 pages (1994).
There is often a requirement for extending the blood circulation half-lifes of pharmaceutical proteins. Short half lives require not only frequent, but also high does for therapeutic effect. Extending the half life permits lower, less frequent and thus potentially safer doses which are also cheaper to produce.
Even the most promising protein therapeutic will also not be beneficial if its stability cannot be maintained during packaging, shipping, sotrage and administration. Carpenter “Inhibition of Stress-Induced Aggregation of Protein Therapeutics” Methods in Enzymology, 309, 1999, discusses types of stresses and conditions that are routinely found to cause aggregation of purified therapeutic proteins.
For mAbs to be viable therapeutics, they msut be formulated to have low viscosity, be chemically stable and have normal in vivo clearance rates. There is significant correlations with simple physical properites obtaininable from antibody sequences and molecular dynamics simulations. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearnace correlates with high hydrophobicities of certain complementarity determinign regions and with high positive or high hegative net charge. Chemical degradation from tyrptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerizaiton rates can be predicted from solvent exposure and flexibility as determeind by molecular dynamics simulations. mabs differing largely in the CDR sequence or in the Fv domain exhibit a variety of viscosity concetnraiton profiles under similar conditions of shear ate. (Sharma “In silico selection of therapeutic antibodies for development: viscosity, clearance, and chemical stability” PNAS, 111(52), 2014).
Zhou (US 16/998,391, published as US 20210054050) disclsoes optimizing formulation of bispecific antibodies whih incldues determining determing a profile of protein-protein interactions pysico-chemical properites such as isoelectric point, surface hydrophobicity, surface cahrge, viscosity and of the bispecific antibody and optimizing or selecting at least one component of the formulation based on the profile of the protein-protein interactions. Molecular condidates for constructing bispecific antibodies such as selecting a combination of ppeptides or proteins to produce bispecific antibodies based ont he profile of the protein-protein interaction and/or the profile of the physico-chemical properties of teh peptdies or proteins having desired amino acid sequences is also provdied. Methods for characterizing protein-protein interactions include dynamic light scattering (DLS), static light scattering (SLS). In DLS measurement, the nature and magnitude of protein-prtoein itneractions can be extrapolated as interaction parameter kD from the non-ideal dependence of diffusion coefficient on protein concetnraiton in the relatively diluted regime. For example, comapring a BsAb1, the itneraction parameter KD of mAb-A had a much larger negative value in teh presence of 10 mM histidine at pH 6, incidcating the presence of much strong attractice protein-protein itneractions. Creasing ionic strengh by adding 150 mM NaCl or 150 mM ArgHCl and chaingin pH value (from pH 8 to pH 5) can increase the KD values of teh mAb-A formualtions. Thus, increasing ionic strengh, lowering pH value and using hydrophobic exicpient caneffectively reduce attractice prtoein-protein interaction.
Types of Instability and Methods used to Counteract
Aggregation: Protein aggregation is a term that includes many types of molecular assemblies. Aggregation can arise from non-covalent interactions or from covalently linked species, which can vary widely in terms of reversibility. Aggregation can be cause by incorrect folding during protein expression and perturbation of the native conformation during protein purificaiton, formulation, freez drying. (Manning, Pharmaceutical Research, 27(4), 2010)
Deamidation: which involves the hydrolysis of Asn and Gln side chain amides is a common degradation pathway for proteins and peptides. A number of formulation approahces have been described to slow deamidation. The most effective approach is to control pH. Deamidation for a single reactive Asn displays a V shaped pH rate profile, withi the minimum being between pH 3 and 6. (Manning, Pharmaceutical Research, 27(4), 2010).
Gradual deamidation of amino acids, such as asparagine, is a cause of protein instability. In the deamidation reaction, the side chain amid of a Gln or Asn residue is hydrolzed to form a free carboxylic acid (Pharmaceutical Research, 7(7), 1990). The substitution of asparagine with another amino acid by site directed mutagenesis is considered the most certain method to prevent deamidation of proteins (sugo, 2005/0171339).
Denaturation: denotes the loss of the globular or three dimensioanl structure that most proteins adopt. Probably the most common stress that casues globular struture of proteins to be lost is elevated termpature. (Manning, Pharmaceutical Research, 27(4), 2010)
Oxidation: Any protein that contains His, Met, Cys, Tyr and Trp amino acids can be potentially damaged by reaction with any of a number of reactive oxygen species (ROS). A number of approahces can limit oxidation of proteins such as minimizing exposure to oxygen by reducing the headspace in the vial. Sugars adn polyols can complex metals at high concentraiton, thereby reducing MCO induced damage. (Manning, Pharmaceutical Research, 27(4), 2010)
Techniques used to Increase Protein Half-lives (As to PEG -see outline)
A key element in drug development is to achieve adequate circulating half-lives, which impact dosing, drug adminsitraiton and efficacy. Many approaches have been understaken with teh aim to increase the half-life of biotherpaeutics including PEGylation, conjugation or genetic fusion with proteins (e.g., transferrin, albumin, growth hormone), conjugation or fusion with Fc fragmetns, glycosylation and mutagenesis. (Roopenian, WO 2009/097017)
Biotinylation of Fc domain:
Roopenian (WO2009/097017) discloses that biotinylation of the Fc domain of an antibody increases the serum half life of the antibody. In one embodiment, the bitoin moeity is coavlently conjugated to the CH2-CH3-hinge region to a solvent exposed lsyine within the human Fc domain. In certain embodiments biotinylation can be acheived by utilizing the E. coli biotin holoenzyme synthetase. in other embodiments, biotin can be coupled to primary and epsilon amines of lysine either direclty or via a spacer using for example the N-hydroxysuccinimide (NHS) ester chemistry. An exemplary method is to label the epsilon amino groups of lysine residues with duccinimidyl ester of biotein. In other embdoiments, the Fc fragment may be mutagenized such that only 1, 2 or 3 lysine residues are available for crosslinking to a biotin moeity. For example, ylsines not used for biotin conjugation can be mutagenized toa lanine. Exemplary lusine reidues which may be conjugated include K246, K274, K288, K317, K320 of the IgHG1 Fc fragment.
Techniques to Prevent Aggregation:
Gurny (WO 2011/121560) disclsoes a method fo stabilizing an antibody by decreasing its aggregation propensity bymasking at least one specific residue from the CH domain of the Fc region. In particular, aggregation could be modulated by blocking or masking at least one of the lysine resiudes corresponding to Lys445B and Lys383B of an IgG1 crystal stru ture on teh Fc region. To do this a modulator compound (adenosine 5′ monophosphate) that interacts preferentially with the lysine residue in the CH domain of the antibody (bevacizumab) was identified.
Techniques used to Prevent Oxidation
Numerous processes and additives are known for the stabilization of proteins in solution. The prevention of protein oxidation in pharamceutical formulations generally employs strategies such as reduction in temperature, protection from light, cahnge in formulation pH, removal of oxygen from the head space, removal of excipients that have contaminants that catalyze oxidation (such as polysorbates) and addition of antioxidates. (Cleland, Critical Reviews in Therapeutic Drug Carrier System, 10(4): 307-377 (1993)
Amino Acids:
All amino acids are susceptible to oxidation, although their susceptibilities vary greatly. Organisms have evolved complex antioxidant defenses to minimize oxidative damage and repair systems for reversing some oxidative modificationsas well as disposal systems for removing modified macromolecules that are not repaired. Oxidative modification of residues within proteins may be mediated by a variety of systems including oxidasees, ozone, hydrogen peroxide, superoxide, gamma-irradiation, metal catalyzed oxidation, “leakage” from the elctron transport chain and auto-oxidation of flavins or xenobiotics. (Levine, Proc. Natl. Acad. Sci. USA, 93, 1996, 15036-15040)
–Methionine:
Cry (US 2003/0103895A1) discloses peptides such as antibodies may be stored longer by the addition of a hydrophilic thioether such as methionine and a hydrophilic 6-hydroxy-chroman derivative such as vitamine E.
Hora (WO 90/00397) discloses compositions for proteins which are not stably soluble such as IL-2. The composition includes amino acids, vitamins, polymers, fatty acids and low molecular weight acids. Specifically, the IL-2 stabilizer is selected amoung carnitine, carnitine, betaine, pyridoxine, salts of capric acid or succinic acid polyvinylpyrrolidone.
Takruri (WO 92/15614) disclsoes a method for inhibiting the oxidation of a polypeptide having an amino acid sequence comprising at least one methionine by adding methionine.
Fusion to Fc fragments: Researches have increased protien half life by fusion to Fc fragments (WO 00/24782)
Freezing: In general, protein stability can be increased by cooling or freezing, although there have been exceptions. (Cleland, Critical Reviews in Therapeutic Drug Carrier System, 10(4): 307-377 (1993)
Effect of pH
The mechanism of deamidation often dictates the best choice of pH. Lowering the pH in r-met hGH reduced the rate of deamidation but induced particulate formation. The presence of Asp-Gly sequences also make controlling deamidation by lowering pH more difficult. The rate of degradation of Asp-contianing hexapeptide incdreases near pH 3 to 5.
Stabilization by Drying: Since deamidation is a hydrolysis reaction that requires water, an obvious strategy to decrease deamidation is to eliminate water and one of the most useful techniques for water removal is feeze drying (“lyophilization”). (Cleland, Critical Reviews in Therapeutic Drug Carrier System, 10(4): 307-377 (1993)
Despite the icnreased cost of production of lyophilizaiton, thsi process can often provide advantages for shipping, long term storage stability and improved stability to temperature. (Manning, Pharmaceutical Research, 27(4), 2010)
Strategies to increase protein stability in packaging and shipment
Proteins, such as antibodies, are often generated and stored for later use. It is important that such proteins be stored under conditions that preserve the stability of the protein.
While liquid antibody formulations are less expensive, faster to develop and generally easier to prepare for administraiton than alternative formulation approaches, liquid antibody formulations are prone to oxidation, deamidation, aggregation and fragmentation. Lyophilization or introduction into hydrophobic polymer systems can reduce the impact of water on antibody drug formulations. (Dougherty, Advanced Drug Delivery Reviews 58 (2006) 686-706).
Lyophilization or freeze drying in pharmaceutical manufacture is a process in which a pure dissolved product is frozen and then dried by exposure to conditions that cause sublimation of the ice. Lyophilization, like crystallization, is a useful final stage procedure in biomanufacturing becasue it provides a way to prevent the reactions that normally occur in solution. Therefore, the process can enhance the stability of a protin pharmaceutical and allow it to be stored for prolonged periods at ambient temperatures without fear of degradation or loss of activity. Lyophilization is relatively expensive to carry out for large scale processes but the benefits of increasing drug longevity and eliminating the requirement for a cold chain can far outweight the initial costs. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)
Formulation Excipients:
Proteins generally reuqire a formulation excipient(s) as a protein stabilizer in a liquid state. Protein stabilizaton by a stabilizer(s) can be achieved through the traditional preferential interaction mechanism and/or other proposed mechanisms such as nonspecific interaction with surface hydrophobic pockets or charged amino acids, specific ligand binding, and enhancement of solution fiscosity. To enhance the stability of proteins, simultaneous use of multiple stabilizers has been tested in expectation of addressing different stability issues via different mechanisms and/or possible synergistic effect. For example, a mixture of three amino acids; L-arginine (positively charged), L-glutamic acid (negatively charged) and L-isoleucine (nonpolar) can stabilize recombinant factor VIII during lyophiliation and storage to the same degree as achieved by using albumin as a stabilizing excipient. (Wang “Advanced protein formulations” Protein Science 24: 1031-1039, 2015)
PhH Conditions: Li (WO 2006/020935) described formualtions that are suitable for stroage of proteins over a relatively borad range of protein concentrations, pH and buffer types.
Polar Organic Solvents:
Generally, addition of a miscible oranic solvent into an aqueous protein solution makes the protein unstable due to promotion of protein unfolding. On the other hand, changing the solvent property could lead to a reduction in protein solution viscosity. Polar organic solvents have been evaluated for this purpose. The viscosity of an IgG1 solution was reduced significantly in the presence of a small amount of diemthyl sulfoxide (DMSO) or dimethylacetamide (DMA). The effect is roughly equilvaent to addition of 200 mM arginine chloride. However, the three melting temperatures of the protein dropped linearly with increasing concentraitons of these two solvents. (Wang “Advanced protein formulations” Protein Science 24: 1031-1039, 2015)
Freeze drying (lyophilization) is considered useful and effective for preservation of many biologically active materials, including proteins (see US 6020469). Studies have shown that freeze drying antibodies to a specific percent of residual water (usually 1-8%) allows for optimal stabilization in the drug state and stability druing reconstitution. (Dougherty, Advanced Drug Delivery Reviews 58 (2006) 686-706).
Lyophilization or freeze-drying in pharmaceutical manufacture is a process in which a pure dissolved product is frozen and then dried by exposure to conditions that cause sublimation of the ice.
In reality, use of a freeze drying process to acheive room temperature stabiliyt for proteins is not ideal due to the added manufactuing cost (long feeze drying prcoess) and the extra reconstitution step need for drug adminsitration. (Wang “Advanced protein formulations” Protein Science 24: 1031-1039, 2015)
–Plate based: Plate-based freezing and thawing is one method of freezing biopharmaetuical rpdocuts. Plate freezers such as single use Support’s RoSS.pFTU system ensure controlled and uniform freeqing, helping to prevent products form degrading druing the prcoess. Compared with spray drying and conventional freezing technologies, optimized en-to-end cold-chain namagemetn helps biomanufacturers reduce product lossses. (Daniel Tischler, “Frrezing can replace spray drying for builk intermediates” BioProcess International, June 2023, 21(6).
Site-Specific Mutagenesis:
Site-specific mutagenesis can be very effective for protein stabilization. For example, a lipase mutant increased the Tm of the wild type protein by as much as 22C (from 78C) and the optimum activity temperature by as much as 30C, devoid of heat induced aggregation. The melting temeprature of a mutated RNase Sa was 28C higher than the wild type enzume. Mutation of even one amino acid at a key position may later the stability of a protein. (Wang “Advanced protein formulations” Protein Science 24: 1031-1039, 2015)
Chemical Conjugation/Protein Fusion:
chemical conjugation or protein fusion was initially designed as a strategy to increase the in vivo half-life for longer duration of action. An added potential benefit upon chemical conjugation or protein fusion is to enahnce protein stability due to steric hindrance and/or changes in surface properties. Conjugaiton moieties include PEG, glycans and other hydrophophilic substances. (Wang “Advanced protein formulations” Protein Science 24: 1031-1039, 2015)
Whole Blood:
While Blood is collected form donors for the preparation of plasma and cell concentrates. After collection, whole blood can be sotred under different condtions: at room termpature for up to 24h as awell as 1-6C for a maxium of 15 h.
Container Materials:
Containers and vessels used during manufacturing are single use disposable componets or metal tanks, primarily either 316L stainless steel (SS) of Hastelloy nickel-based allowy (HLY) (trademark of Haynes International). Bedi “Container Materials in Biopharmaceuticals” BioProcess Intertional, November-December 2022, reported the relative inert natural of HLY compared with SS with respect to its effects on product quality (speifically, the impact on oxidation) of two MAbs.
As to Particular Types of Proteins
HSA:
Human blood serum albumin (HSA) conjugates: Researches have increased protein half life by function is HSA (5,766,883). Edwards (US 2006/0141456) discloses an interesting way to increase stability along these lines by selecting peptide sequences that selectively bind to HSA with high affinity in most tissues but interacts only weakly with HSA in a target tissue. As a consequence, such a peptide will be transported with high efficiency and long slow clearance through the blood stream and will be selectively released in the target tissue. Of particular interest were agents that bind to HSA with high affinity at normal pH (about 7.4) but with weaker affinity at lower pH.
Site-Directed Mutagenesis: Mutagenesis can be used to improve the solubility of the protein.
Immuoglobulins: See outline