An Industry Perspective on the use of Forced Degradation Studies to Assess Comparability of Biopharmaceuticals.

Forced degradation, also known as stress testing, is used throughout pharmaceutical development for many purposes including assessing the comparability of biopharmaceutical products according to ICH Guideline Q5E. These formal comparability studies, the results of which are submitted to health authorities, investigate potential impacts of manufacturing process changes on the quality, safety, and efficacy of the drug. Despite the wide use of forced degradation in comparability assessments, detailed guidance on the design and interpretation of such studies is scarce. The BioPhorum Development Group is an industry-wide consortium enabling networking and sharing of common practices for the development of biopharmaceuticals. The BioPhorum Development Group Forced Degradation Workstream recently conducted several group discussions and a benchmarking survey to understand current industry approaches for the use of forced degradation studies to assess comparability of protein-based biopharmaceuticals. The results provide insight into the design of forced degradation studies, analytical characterization and testing strategies, data evaluation criteria, as well as some considerations and differences for non-platform modalities (e.g., non-traditional mAbs). This article presents survey responses from several global companies of various sizes and provides an industry perspective and experience regarding the practicalities of using forced degradation to assess comparability.

Kinetic mechanism of phenylalanine hydroxylase: intrinsic binding and rate constants from single-turnover experiments.

Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH(4)) and O(2). A complete kinetic mechanism for PheH was determined by global analysis of single-turnover data in the reaction of PheHΔ117, a truncated form of the enzyme lacking the N-terminal regulatory domain. Formation of the productive PheHΔ117-BH(4)-phenylalanine complex begins with the rapid binding of BH(4) (K(d) = 65 µM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (K(d) = 130 µM) is approximately 10-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O(2) rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, which can be detected as a decrease in absorbance at 340 nm, with a rate constant of 140 s(-1). Formation of the 4a-hydroxypterin and Fe(IV)O intermediates is 10-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines k(cat). Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation.

Single turnover kinetics of tryptophan hydroxylase: evidence for a new intermediate in the reaction of the aromatic amino acid hydroxylases.

Tryptophan hydroxylase (TrpH) uses a non-heme mononuclear iron center to catalyze the tetrahydropterin-dependent hydroxylation of tryptophan to 5-hydroxytryptophan. The reactions of the TrpH.Fe(II), TrpH.Fe(II).tryptophan, TrpH.Fe(II).6MePH(4).tryptophan, and TrpH.Fe(II).6MePH(4).phenylalanine complexes with O(2) were monitored by stopped-flow absorbance spectroscopy and rapid quench methods. The second-order rate constant for the oxidation of TrpH.Fe(II) has a value of 104 M(-1) s(-1) irrespective of the presence of tryptophan. Stopped-flow absorbance analyses of the reaction of the TrpH.Fe(II).6MePH(4).tryptophan complex with oxygen are consistent with the initial step being reversible binding of oxygen, followed by the formation with a rate constant of 65 s(-1) of an intermediate I that has maximal absorbance at 420 nm. The rate constant for decay of I, 4.4 s(-1), matches that for formation of the 4a-hydroxypterin product monitored at 248 nm. Chemical-quench analyses show that 5-hydroxytryptophan forms with a rate constant of 1.3 s(-1) and that overall turnover is limited by a subsequent slow step, presumably product release, with a rate constant of 0.2 s(-1). All of the data with tryptophan as substrate can be described by a five-step mechanism. In contrast, with phenylalanine as substrate, the reaction can be described by three steps: a second-order reaction with oxygen to form I, decay of I as tyrosine forms, and slow product release.

Demonstration of a peroxide shunt in the tetrahydropterin-dependent aromatic amino acid monooxygenases.

The nonheme iron enzyme phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase catalyze the hydroxylation of their aromatic amino acid substrates using a tetrahydropterin as the source of electrons. The hydroxylating intermediate is proposed to be an Fe(IV)O species. We report here that all three enzymes will catalyze hydroxylation reactions using H(2)O(2) in place of tetrahydropterin and oxygen, forming tyrosine and 3-hydroxyphenylalanine from phenylalanine, 4-HOCH(2)-phenylalanine from 4-CH(3)-phenylalanine, and hydroxycyclohexylalanine from 3-cyclohexylalanine. No peroxide-dependent reaction is seen with active site mutants of TyrH and PheH in which the stability or reactivity of the iron center is compromised. These results provide further support for an Fe(IV)O hydroxylating intermediate.

Insights into the catalytic mechanisms of phenylalanine and tryptophan hydroxylase from kinetic isotope effects on aromatic hydroxylation.

Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-(2)H]-, [3,5-(2)H(2)]-, and (2)H(5)-phenylalanine as substrates. All (D)k(cat) values are normal with Delta117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12-1.41. In contrast, for Delta117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the (D)k(cat) value with [4-(2)H]-phenylalanine is 0.92 but is normal with [3,5-(2)H(2)]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Delta117PheH V379D shows a similar inverse isotope effect with [4-(2)H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-(2)H]-and [3,5(2)H(2)]-phenylalanine, are identical for Delta117PheH and Delta117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the (D)k(cat) value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight ( approximately 34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With (2)H(5)-indole-tryptophan as a substrate for Delta117PheH, the (D)k(cat) value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the (D)k(cat) values are within error of those for Delta117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine.

Intrinsic isotope effects on benzylic hydroxylation by the aromatic amino acid hydroxylases: evidence for hydrogen tunneling, coupled motion, and similar reactivities.

Deuterium kinetic isotope effects for hydroxylation of the methyl group of 4-methylphenylalanine have been used as a probe of the relative reactivities of the hydroxylating intermediates in the aromatic amino acid hydroxylases phenylalanine, tyrosine, and tryptophan hydroxylase. When there are three deuterium atoms in the methyl group, all three enzymes exhibit an intrinsic isotope effect of about 13. The temperature dependence of the isotope effect is consistent with moderate tunneling, with the extent of tunneling identical for all three enzymes. In the case of phenylalanine hydroxylase, the presence of the regulatory domain has no effect on the values. The intrinsic primary and secondary isotope effects were determined using 4-methylphenylalanine containing one or two deuterium atoms in the methyl group. With one deuterium atom, the intrinsic primary and secondary effects have average values of 10 and 1.1, respectively. With two deuterium atoms, the primary effects decrease to 7.4 and the secondary effect increases to 1.3, consistent with coupled motion of the primary and secondary hydrogens. The results with all three enzymes are consistent with a hydrogen abstraction mechanism. The similarities of the isotope effects and extent of tunneling establish that the reactivities of the hydroxylating intermediates in the three enzymes are essentially identical.